Methods and systems for droplet-based single cell barcoding

ABSTRACT

Methods and systems are provided for sample preparation techniques and sequencing of macromolecular constituents of cells and other biological materials.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/887,947, filed Feb. 2, 2018, which is a continuation of PCTApplication No. PCT/US2018/16019, filed Jan. 30, 2018, which claimspriority to U.S. Provisional Patent Application No. 62/452,261, filedJan. 30, 2017, U.S. Provisional Patent Application No. 62/500,943, filedMay 3, 2017, and U.S. Provisional Patent Application No. 62/570,783,filed Oct. 11, 2017, each of which is entirely incorporated herein byreference for all purposes.

BACKGROUND

Whole genome amplification and sequencing technologies are beginning tofind broader adoption. These technologies may not consider theheterogeneity of a sample; instead, they may assume that all species tobe amplified or sequenced come from a homogeneous population of cells orother biological materials (such as viruses). However, certainapplications may benefit from the amplification or sequencing of speciesobtained from single cells obtained from a much larger population. Insome cases, the single cells of interest may be quite rare. Forinstance, cancerous cells may undergo continuous mutations in theirdeoxyribonucleic acid (DNA) sequences. Cancer researchers or oncologistsmay wish to amplify and sequence the genomes of such cells or of otherindividual cells. They may find, however, that sequencing dataattributable to the single cells of interest is obscured by that arisingfrom far more prevalent cells. Thus, there is a need for samplepreparation techniques that allow partial or whole genome amplificationand sequencing of single cells of interest.

SUMMARY

Provided herein are methods and systems for sample preparationtechniques that allow amplification (e.g., whole genome amplification,reverse transcription, amplification of cellular nucleic acids, etc.)and sequencing of single cells, which may be of interest. The methodsand systems generally operate by bringing together a first liquid phasecomprising a plurality of biological particles (e.g., particlescomprising a cell or a cell component(s)), a second liquid phasecomprising gel beads, and a third immiscible phase. The liquid phasesmay interact to form partitions (e.g., droplets). Some of the partitionsmay contain a single biological particle or a plurality of biologicalparticles and one or more gel beads. The methods and systems may beconfigured to allow the implementation of a single operation ormulti-operation chemical and/or biochemical processing within thepartitions.

Methods and systems of the present disclosure may allow particularbiochemical operations to occur in a droplet prior to allowing otherbiochemical operations to occur in the droplet. The droplet may containa gel bead which may contain a tag (such as a barcode) that may be usedto barcode macromolecular constituents (e.g., nucleic acid molecules) ofa single biological particle.

Methods and systems of the present disclosure may be used to generatetarget sequence or sequencing reads (“reads”) specific to macromolecularconstituents of interest at a higher rate than non-target specificreads. For instance, the methods and systems are characterized by theirsuppression of no template control (NTC) effects.

In an aspect, the present disclosure provides a method for analysis of asingle biological particle, comprising (a) providing a first liquidphase comprising a plurality of biological particles; (b) providing asecond liquid phase comprising a plurality of beads each including a tagto barcode one or more macromolecular constituents of each of theplurality of biological particles; (c) bringing the plurality ofbiological particles from the first liquid phase and the plurality ofbeads from the second liquid phase in contact with a third liquid phasethat is immiscible with the first or second liquid phase, to partitioneach of the plurality of biological particles and the plurality of beadsinto a plurality of partitions (e.g., droplets), wherein uponpartitioning, a given partition of the plurality of partitions includesa single biological particle from the plurality of biological particlesand a single bead from the plurality of beads; (d) in the givenpartition (e.g., droplet), using the tag from the single bead to barcodethe one or more macromolecular constituents of the single biologicalparticle, forming one or more barcoded macromolecules; and (e)subjecting the barcoded macromolecules to sequencing to generate readscharacterized by a specific target read(s) to non-target specificread(s) ratio greater than 1, which specific target read(s) of the readsis indicative of the one or more macromolecular constituents.

In some embodiments, the sequencing is nucleic acid sequencing. In someembodiments, the nucleic acid sequencing is massively parallelsequencing. In some embodiments, the nucleic acid sequencing is digitalpolymerase chain reaction (PCR).

In some embodiments, the specific target read(s) to non-target specificread(s) ratio is greater than 100. In some embodiments, the specifictarget read(s) to non-target specific read(s) ratio is greater than1,000. In some embodiments, the specific target read(s) to non-targetspecific read(s) ratio is greater than 10,000. In some embodiments, thespecific target read(s) to non-target specific read(s) ratio is greaterthan 100,000. In some embodiments, the specific target read(s) tonon-target specific read(s) ratio is greater than 1,000,000. In someembodiments, the specific target read(s) to non-target specific read(s)ratio is greater than 10,000,000. In some embodiments, the specifictarget read(s) to non-target specific read(s) ratio is greater than100,000,000. In some embodiments, the specific target read(s) tonon-target specific read(s) ratio is greater than 1,000,000,000.

In some embodiments, the specific target read(s) correspond to one ormore nucleic acid sequences from the single biological particle. In someembodiments, the non-target specific read(s) corresponds to one or moreexogenous nucleic acid sequences.

In some embodiments, the plurality of partitions is a plurality ofdroplets. In some embodiments, the plurality of partitions is aplurality of wells.

In some embodiments, a given bead of the plurality of beads includes oneor more tags coupled to a surface thereof and/or enclosed within thegiven bead.

In some embodiments, the plurality of partitions is part of a populationof partitions that includes one or more partitions that are unoccupiedby biological particles and/or beads.

In another aspect, the present disclosure provides a method for analysisof a single biological particle, comprising (a) providing a first liquidphase comprising a plurality of biological particles; (b) providing asecond liquid phase comprising a plurality of beads each including a tagto barcode one or more macromolecular constituents of each of theplurality of biological particles; and (c) bringing the plurality ofbiological particles from the first liquid phase and the plurality ofbeads from the second liquid phase in contact with a third liquid phasethat is immiscible with the first or second liquid phase, to partitioneach of the plurality of biological particles and the plurality of beadsinto a plurality of partitions, wherein upon partitioning, a givenpartition of the plurality of partitions includes a single biologicalparticle from the plurality of biological particles and a single beadfrom the plurality of beads, wherein the single biological particleincludes or is enclosed within a polymer or gel matrix.

In some embodiments, the first liquid phase further comprises precursorsthat are capable of being polymerized or gelled. In some embodiments,the method comprises subjecting the first liquid phase to conditionssufficient to polymerize or gel the precursors so as to encapsulate thesingle biological particle in the polymer or gel matrix. In someembodiments, the polymer or gel matrix is diffusively permeable toreagents while retaining the one or more macromolecular constituents.

In some embodiments, the method comprises subjecting the singlebiological particle to conditions sufficient to lyse the singlebiological particle to provide a lysed single biological particle. Insome embodiments, the method comprises subjecting the lysed singlebiological particle to conditions sufficient to denature the one or moremacromolecular constituents released from the lysed single biologicalparticle. In some embodiments, the method comprises subjecting the lysedsingle biological particle to conditions sufficient to release the oneor more macromolecular constituents from the polymer or gel matrix.

In some embodiments, the method comprises using the tag from the singlebead to barcode the one or more macromolecular constituents, forming oneor more barcoded macromolecules. In some embodiments, the methodcomprises subjecting the barcoded macromolecules to sequencing.

In some embodiments, the polymer or gel matrix includes one or more ofdisulfide crosslinked polyacrylamide, agarose, alginate, polyvinylalcohol, PEG-diacrylate, PEG-acrylate/thiol, PEG-azide/alkyne, otheracrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, andelastin.

In some embodiments, the conditions sufficient to lyse the singlebiological particle comprises exposure to sodium hydroxide (NaOH). Insome embodiments, the conditions sufficient to denature the one or moremacromolecular constituents comprises exposure to sodium hydroxide(NaOH). In some embodiments, the conditions sufficient to release theone or more macromolecular constituents comprises exposure todithiothreitol (DTT). In some embodiments, the one or moremacromolecular constituents released from the lysed single biologicalparticle are denatured prior to (c).

In some embodiments, the sequencing is nucleic acid sequencing. In someembodiments, the nucleic acid sequencing is massively parallelsequencing. In some embodiments, the nucleic acid sequencing is digitalpolymerase chain reaction (PCR).

In some embodiments, the third liquid phase includes an oil. In someembodiments, the oil includes a fluorinated hydrocarbon. In someembodiments, the first liquid phase and the second liquid phase are thesame phase.

In some embodiments, the first liquid phase and the second liquid phaseare mixed to provide a mixed phase, and the mixed phase is brought incontact with the oil phase.

In some embodiments, the single biological particle comprises anorganelle. In some embodiments, the single biological particle comprisesa virus. In some embodiments, the single biological particle comprises acell. In some embodiments, the cell comprises a rare cell from apopulation of cells.

In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10² cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10³ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10⁴ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10⁵ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10⁶ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10⁷ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10⁸ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10⁹ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10¹⁰ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10¹¹ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10¹² cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10¹³ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10¹⁴ cells of the population ofcells. In some embodiments, the rare cell is present in a sample at aconcentration of at least about 1 in 10¹⁵ cells of the population ofcells.

In some embodiments, the rare cell is a cancerous cell. In someembodiments, the cancer cell is a circulating tumor cell. In someembodiments, the rare cell is a cell obtained from an in vitrofertilization procedure. In some embodiments, the rare cell is a cellobtained from an individual displaying genetic mosaicism. In someembodiments, the rare cell is a cell obtained from an organism producedusing synthetic biology techniques. In some embodiments, the populationof cells is a heterogeneous population of cells.

In some embodiments, the method comprises obtaining the plurality ofbiological particles. In some embodiments, the plurality of biologicalparticles is obtained from a blood of a subject. In some embodiments,the plurality of biological particles includes cells. In someembodiments, the cells are cancerous cells. In some embodiments, theplurality of biological particles is obtained from a tissue of asubject.

In some embodiments, the one or more macromolecular constituentscomprise deoxyribonucleic acid (DNA). In some embodiments, the one ormore macromolecular constituents comprise ribonucleic acid (RNA). Insome embodiments, the one or more macromolecular constituents comprisepeptides or proteins.

In some embodiments, the tag is a primer. In some embodiments, (d)further comprises subjecting single biological particle to conditionssufficient for nucleic acid amplification. In some embodiments, theconditions sufficient for nucleic acid amplification comprise primingfree amplification. In some embodiments, the priming free amplificationcomprises priming free amplification by polymerization at nick sites.

In some embodiments, the method further comprises using the tag toidentify the one or more macromolecular constituents of the singlebiological particle from the plurality of biological particles. In someembodiments, the method further comprises subjecting the barcodedmacromolecules to nucleic acid sequencing to identify the one or moremacromolecular constituents. In some embodiments, the nucleic acidsequencing is untargeted sequencing. In some embodiments, the nucleicacid sequencing is targeted sequencing.

In some embodiments, the plurality of partitions is a plurality ofdroplets. In some embodiments, the plurality of partitions is aplurality of wells.

In some embodiments, a given bead of the plurality of beads includes oneor more tags coupled to a surface thereof and/or enclosed within thegiven bead.

In some embodiments, the plurality of partitions is part of a populationof partitions that includes one or more partitions that are unoccupiedby biological particles and/or beads.

In another aspect, the present disclosure provides a method for analysisof a single biological particle, comprising (a) providing a plurality ofbiological particles, and a plurality of beads each including a tag tobarcode one or more macromolecular constituents of each of the pluralityof biological particles; and (b) partitioning the plurality ofbiological particles and the plurality of beads into a plurality ofpartitions, wherein upon partitioning, a given partition of theplurality of partitions includes a single biological particle from theplurality of biological particles and a single bead from the pluralityof beads, wherein the single biological particle includes or is enclosedwithin a gel or polymer matrix within the given partition.

In some embodiments, the plurality of partitions is a plurality ofdroplets. In some embodiments, the plurality of partitions is aplurality of wells.

In some embodiments, a given bead of the plurality of beads includes oneor more tags coupled to a surface thereof and/or enclosed within thegiven bead.

In some embodiments, the plurality of partitions is part of a populationof partitions that includes one or more partitions that are unoccupiedby biological particles and/or beads.

In another aspect, the present disclosure provides a system for analysisof a single biological particle, comprising a partition generatorcomprising (i) a first source of a first liquid phase comprising aplurality of biological particles, (ii) a second source of a secondliquid phase comprising a plurality of beads each including a tag tobarcode one or more macromolecular constituents of each of the pluralityof biological particles, and (iii) a third source of a third liquidphase that is immiscible with the first or second liquid phase; and acontroller operatively coupled to the partition generator, wherein thecontroller is programmed to (i) bring the first liquid phase from thefirst source and the second liquid phase from the second source incontact with the third liquid phase from the third source along a firstchannel to partition each of the plurality of biological particles andthe plurality of beads into a plurality of partitions that flow along asecond channel, wherein upon partitioning, a given partition of theplurality of partitions includes a single biological particle from theplurality of biological particles and a single bead from the pluralityof beads; and(ii) in the given partition, use the tag from the singlebead to barcode the one or more macromolecular constituents of thesingle biological particle, forming one or more barcoded macromolecules;and (iii) subject the barcoded macromolecules to sequencing to generatereads characterized by a specific target read(s) to non-target specificread(s) ratio greater than 1, which specific target read(s) of the readsis indicative of the one or more macromolecular constituents.

In another aspect, the present disclosure provides a system for analysisof a single biological particle, comprising a partition generatorcomprising (i) a first source of a first liquid phase comprising aplurality of biological particles, (ii) a second source of a secondliquid phase comprising a plurality of beads each including a tag tobarcode one or more macromolecular constituents of each of the pluralityof biological particles, and (iii) a third source of a third liquidphase that is immiscible with the first or second liquid phase, whereinthe first liquid phase further comprises precursors that are capable ofbeing polymerized or gelled; and a controller operatively coupled to thepartition generator, wherein the controller is programmed to bring theplurality of biological particles from the first liquid phase and theplurality of beads from the second liquid phase in contact with thethird liquid phase that is immiscible with the first or second liquidphase, to partition each of the plurality of biological particles andthe plurality of beads into a plurality of partitions, wherein uponpartitioning, a given partition of the plurality of partitions includesa single biological particle from the plurality of biological particlesand a single bead from the plurality of beads, wherein the singlebiological particle includes or is enclosed within a polymer or gelmatrix.

In some embodiments, the third liquid phase includes an oil. In someembodiments, the first liquid phase and the second liquid phase are thesame phase.

In some embodiments, the plurality of biological particles includescells. In some embodiments, the plurality of biological particles isobtained from a tissue of a subject.

In some embodiments, the one or more macromolecular constituentscomprise deoxyribonucleic acid (DNA). In some embodiments, the one ormore macromolecular constituents comprise ribonucleic acid (RNA). Insome embodiments, the tag is a primer.

In some embodiments, the controller subjects the single biologicalparticle to conditions sufficient for nucleic acid amplification. Insome embodiments, the controller is programmed to subject the singlebiological particle to conditions sufficient to barcode at least onemacromolecular constituent from the single biological particle with atleast one tag from the single bead.

In another aspect, the present disclosure provides a non-transitorycomputer-readable medium comprising machine-executable code that, uponexecution by one or more computer processors, implements a method foranalysis of a single biological particle, the method comprising (a)providing a first liquid phase comprising a plurality of biologicalparticles; (b) providing a second liquid phase comprising a plurality ofbeads each including a tag to barcode one or more macromolecularconstituents of each of the plurality of biological particles; (c)bringing the plurality of biological particles from the first liquidphase and the plurality of beads from the second liquid phase in contactwith a third liquid phase that is immiscible with the first or secondliquid phase, to partition each of the plurality of biological particlesand the plurality of beads into a plurality of partitions, wherein uponpartitioning, a given partition of the plurality of partitions includesa single biological particle from the plurality of biological particlesand a single bead from the plurality of beads, wherein the singlebiological particle includes or is enclosed within a polymer or gelmatrix.

In another aspect, the present disclosure provides a method for cellularanalysis, comprising

-   (a) partitioning a plurality of cells or derivatives thereof into a    plurality of partitions, wherein upon partitioning, a given    partition of the plurality of partitions includes a single cell or    derivative thereof from the plurality of cells or derivatives    thereof and a set of tags that are capable of barcoding one or more    macromolecular constituents of the single cell or derivative    thereof, wherein the single cell or derivative thereof includes or    is enclosed within a gel or polymer matrix within the given    partition; (b) using the set of tags to barcode the one or more    macromolecular constituents from the single cell, thereby providing    one or more barcoded macromolecules; and (c) analyzing the one or    more barcoded macromolecules or derivatives thereof.

In some embodiments, the one or more macromolecular constituents includedeoxyribonucleic acid. In some embodiments, the one or moremacromolecular constituents include ribonucleic acid.

In some embodiments, the plurality of partitions are a plurality ofdroplets. In some embodiments, the plurality of partitions are aplurality of wells. In some embodiments, the set of tags is coupled to abead in the given partition.

In some embodiments, the method further comprises releasing the one ormore barcoded macromolecules or derivatives thereof from the givenpartition prior to analyzing.

In some embodiments, the method further comprises processing the singlecell to include or be enclosed within the gel or polymer matrix prior topartitioning the plurality of cells into the plurality of partitions. Insome embodiments, the method further comprises processing the singlecell to include or be enclosed within the gel or polymer matrix afterpartitioning the plurality of cells into the plurality of partitions. Insome embodiments, the cells are live cells.

In some embodiments, the live cells are capable of being cultured. Insome embodiments, the live cells are capable of being cultured uponenclosure in or when comprising a gel or polymer matrix.

Tags (e.g., barcodes) may be enclosed within the plurality of beads. Asan alternative or in addition to, tags may be coupled to surfaces of theplurality of beads. A given bead may include a plurality of tags.

In another aspect, the disclosure provides a method for processing oranalyzing one or more components from a cell, comprising: (a) providinga plurality of cell beads and a plurality of barcode beads, wherein (i)a cell bead of the plurality of cell beads comprises the one or morecomponents of the cell, which one or more components comprise a nucleicacid molecule, and (ii) a barcode bead of the plurality of barcode beadscomprises a plurality of nucleic acid barcode molecules for barcodingthe nucleic acid molecule; and (b) partitioning the plurality of cellbeads and the plurality of barcode beads into a plurality of partitions,wherein upon partitioning, a partition of the plurality of partitionscomprises the cell bead and the barcode bead.

In some embodiments, the method further comprises performing one or morereactions on the nucleic acid molecule. In some embodiments, the one ormore reactions comprise nucleic acid modification, nucleic acidamplification, nucleic acid insertion, nucleic acid cleavage, reversetranscription, or any combination thereof. In some embodiments, thenucleic acid modification comprises ligation, digestion, methylation,random mutagenesis, bisulfite conversion, uracil hydrolysis, nucleicacid repair, capping, decapping, or any combination thereof. In someembodiments, the nucleic acid amplification comprises isothermalamplification or polymerase chain reaction. In some embodiments, thenucleic acid insertion comprises transposon-mediated insertion,CRISPR/Cas9-mediated insertion, or any combination thereof. In someembodiments, the nucleic acid cleavage comprises transposon-mediatedcleavage, CRISPR/Cas9-mediated cleavage, or any combination thereof. Insome embodiments, the one or more reactions are performed in thepartition. In some embodiments, the one or more reactions are performedoutside the partition. In some embodiments, the one or more reactionsare performed prior to (a). In some embodiments, the one or morereactions are performed subsequent to (a).

In some embodiments, the method further comprises using the plurality ofnucleic acid barcode molecules to generate a barcoded nucleic acidmolecule from the nucleic acid molecule. In some embodiments, generatingthe barcoded nucleic acid molecule comprises nucleic acid amplification.In some embodiments, generating the barcoded nucleic acid moleculecomprises ligation. In some embodiments, the method further comprisesreleasing the barcoded nucleic acid molecule from the partition. In someembodiments, the method further comprises subjecting the barcodednucleic acid molecule or derivative thereof to sequencing. In someembodiments, the method further comprises, prior to the sequencing,subjecting the barcoded nucleic acid molecule or derivative thereof tonucleic acid amplification. In some embodiments, the nucleic acidamplification is isothermal amplification or polymerase chain reaction.In some embodiments, the polymerase chain reaction is digital polymerasechain reaction.

In some embodiments, the cell bead comprises the cell, and the cell beadcomprising the cell is subjected to conditions sufficient to lyse thecell to generate the one or more components. In some embodiments, thecell bead is subject to the conditions sufficient to lyse the cell inthe partition. In some embodiments, the conditions sufficient to lysethe cell comprise exposing the cell beads to a lysis agent. In someembodiments, the conditions sufficient to lyse the cell compriseexposing the cell beads to sodium hydroxide, potassium hydroxide, sodiumdodecyl sulfate, a non-ionic surfactant, a saponin, a proteinase, alytic enzyme, freeze thawing, ultraviolet light, heat, or anycombination thereof. In some embodiments, the non-ionic surfactant is4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100).

In some embodiments, the cell bead includes or is enclosed within a gelor polymer matrix within the partition. In some embodiments, the barcodebead includes or is enclosed within a gel or polymer matrix within thepartition. In some embodiments, the polymer or gel matrix includes oneor more members selected from the group consisting of disulfidecrosslinked polyacrylamide, agarose, alginate, polyvinyl alcohol,PEG-diacrylate, PEG-acrylate/thiol, PEG-azide/alkyne, other acrylates,chitosan, hyaluronic acid, collagen, fibrin, gelatin, and elastin.

In some embodiments, the plurality of partitions is a plurality ofdroplets. In some embodiments, the plurality of partitions is aplurality of wells. In some embodiments, one or more nucleic acidbarcode molecules of the plurality of nucleic acid barcode molecules arecoupled to a surface of the barcode bead and/or enclosed within thebarcode bead.

In some embodiments, the cell bead further comprises additionalreagents. In some embodiments, the partition further comprisesadditional reagents. In some embodiments, the additional reagentscomprise primers, reverse transcriptase enzymes, polymerases,nucleotides, proteases, transposons, endonucleases, switcholigonucleotides, lysis reagents, or any combination thereof. In someembodiments, the nucleic acid molecule is a deoxyribonucleic acidmolecule. In some embodiments, the deoxyribonucleic acid molecule isgenomic deoxyribonucleic acid. In some embodiments, the deoxyribonucleicacid molecule is complementary deoxyribonucleic acid. In someembodiments, the nucleic acid molecule is a ribonucleic acid molecule.In some embodiments, the ribonucleic acid molecule is messengerribonucleic acid. In some embodiments, the method further comprisesrecovering the nucleic acid molecule or a derivative thereof from thepartition.

In some embodiments, the barcode bead is degradable upon application ofa stimulus. In some embodiments, the method further comprises releasingthe plurality of nucleic acid barcode molecules upon application of thestimulus. In some embodiments, the stimulus is a chemical stimulus, abiological stimulus, a temperature change, exposure to light, a pHchange, or any combination thereof. In some embodiments, the chemicalstimulus is a reducing agent. In some embodiments, the reducing agent isdithiothreitol, β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane,tris(2-carboxyethyl) phosphine, or any combination thereof. In someembodiments, the stimulus is a chemical or biological stimulus, and thepartition comprises the stimulus. In some embodiments, the cell bead isdegradable upon application of a stimulus. In some embodiments, thestimulus is a chemical stimulus, a biological stimulus, a temperaturechange, exposure to light, a pH change, or any combination thereof. Insome embodiments, the chemical stimulus is a reducing agent. In someembodiments, the reducing agent is dithiothreitol, β-mercaptoethanol,(2S)-2-amino-1,4-dimercaptobutane, tris(2-carboxyethyl) phosphine, orany combination thereof. In some embodiments, the stimulus is a chemicalor biological stimulus, and the partition comprises the stimulus.

In some embodiments, the plurality of partitions is part of a populationof partitions that includes one or more partitions that are unoccupiedby a cell bead and/or a barcode bead.

In another aspect, the disclosure provides a system for processing oranalyzing one or more components from a cell, comprising: a firstchannel in fluid communication with a first source comprising aplurality of cell beads, wherein a cell bead of the plurality of cellbeads comprises the one or more components of the cell, which one ormore components comprise a nucleic acid molecule; a second channel influid communication with a second source comprising a plurality ofbarcode beads, wherein a barcode bead of the plurality of barcode beadscomprises a plurality of nucleic acid barcode molecules for barcodingthe nucleic acid molecule; and a junction that brings a first phasecomprising the plurality of cell beads from the first channel and theplurality of barcode beads from the second channel in contact with asecond phase that is immiscible with the first phase, to yield aplurality of droplets comprising the plurality of cell beads and theplurality of barcode beads, wherein a droplet of the plurality ofdroplets comprises the cell bead and the barcode bead.

In some embodiments, the first channel and the second channel are thesame channel. In some embodiments, the system further comprises a thirdchannel in fluid communication with a third source comprising additionalreagents, wherein the first phase comprises the additional reagents. Insome embodiments, the system further comprises a fourth channel in fluidcommunication with a fourth source comprising additional reagents,wherein the first phase comprises the additional reagents. In someembodiments, the additional reagents are reagents for nucleic acidamplification, reagents that can degrade or dissolve cell beads and/orbarcode beads, reagents that degrade linkages between barcodes andbarcode beads, or any combination thereof.

Another aspect of the disclosure provides a composition comprising acell bead of a plurality of cell beads and a barcode bead of a pluralityof barcode beads, wherein the cell bead comprises one or more componentsfrom a cell, which one or more components comprise a nucleic acidmolecule, and wherein the barcode bead comprises a plurality of nucleicacid barcode molecules for barcoding the nucleic acid molecule. In someembodiments, the cell bead further comprises additional reagents. Insome embodiments, the additional reagents comprise primers, reversetranscriptase enzymes, polymerases, nucleotides, proteases, transposons,endonucleases, switch oligonucleotides, or any combination thereof. Insome embodiments, the nucleic acid molecule is a deoxyribonucleic acidmolecule. In some embodiments, the deoxyribonucleic acid molecule isgenomic deoxyribonucleic acid. In some embodiments, the deoxyribonucleicacid molecule is complementary deoxyribonucleic acid. In someembodiments, the nucleic acid molecule is a ribonucleic acid molecule.In some embodiments, the ribonucleic acid molecule is messengerribonucleic acid.

In another aspect, the disclosure provides a method for generating acell bead, comprising: (a) providing a plurality of cells and aplurality of polymeric or gel precursors; (b) partitioning the pluralityof cells and the plurality of polymeric or gel precursors into aplurality of partitions, wherein upon partitioning, a partition of theplurality of partitions comprises a cell of the plurality of cells andat least a portion of the polymeric or gel precursors; and (c)subjecting the partitions to conditions suitable for cross-linking orpolymerizing the polymeric or gel precursors to generate the cell bead,wherein the cell bead encapsulates the cell. In some embodiments, themethod further comprises, subsequent to (b), subjecting the cell bead toconditions sufficient to lyse the cell. In some embodiments, theconditions sufficient to lyse the cell comprise exposing the cell beadsto a lysis agent. In some embodiments, the conditions sufficient to lysethe cell comprise exposing the cell beads to sodium hydroxide, potassiumhydroxide, sodium dodecyl sulfate, a non-ionic surfactant, a saponin, aproteinase, a lytic enzyme, freeze thawing, ultraviolet light, heat, orany combination thereof. In some embodiments, the non-ionic surfactantis 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITONX-100),In some embodiments, in (b), the partition comprises a bead. Insome embodiments, the bead is a magnetic bead. In some embodiments, themagnetic bead is a paramagnetic particle.

Another aspect of the present disclosure provides a method forprocessing one or more nucleic acid molecules from a cell, comprising(a) providing a plurality of cells and a plurality of polymeric or gelprecursors; (b) partitioning the plurality of cells and the plurality ofpolymeric or gel precursors into a plurality of partitions, wherein uponpartitioning, a partition of the plurality of partitions comprises (i) anucleic acid molecule, (ii) a cell of the plurality of cells and (iii)at least a portion of the polymeric or gel precursors; (c) subjectingthe plurality of partitions to conditions sufficient to cross-link orpolymerize the polymeric or gel precursors to form a plurality of cellbeads; and (d) partitioning the plurality of cell beads and a pluralityof barcode beads comprising a plurality of nucleic acid barcodemolecules into an additional plurality of partitions, wherein uponpartitioning, a partition of the additional plurality of partitionscomprises the cell bead and the barcode bead. In some embodiments, themethod further comprises, subsequent to (a), subjecting the plurality ofpartitions to conditions sufficient to lyse the plurality of cells,releasing the nucleic acid molecule from the cell into the partition. Insome embodiments, the nucleic acid molecule is a deoxyribonucleic acidmolecule. In some embodiments, the nucleic acid molecule is aribonucleic acid molecule. In some embodiments, in (b), the partitioncomprises a bead. In some embodiments, the bead is a magnetic bead. Insome embodiments, the magnetic bead is a paramagnetic particle. In someembodiments, the method further comprises performing one or morereactions on the nucleic acid molecule. In some embodiments, the methodfurther comprises barcoding the nucleic acid molecule to generate abarcoded nucleic acid molecule. In some embodiments, the method furthercomprises, subsequent to (d), releasing the barcoded nucleic acidmolecule from the partition. In some embodiments, the method furthercomprises subjecting the barcoded nucleic acid molecule or derivativethereof to sequencing.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1A schematically illustrates an example method for generatingdroplets comprising a barcoded bead and a cell bead (e.g., comprising acell or a cell component(s));

FIG. 1B photographically illustrates an example microfluidicarchitecture for generating cell beads;

FIG. 1C photographically illustrates an example microfluidicarchitecture for generating droplets comprising barcoded beads and cellbeads;

FIG. 1D photographically illustrates droplets comprising barcoded beadsand cell beads generated with the architecture shown in FIG. 1C;

FIG. 2 schematically illustrates a microfluidic channel structure forpartitioning individual or small groups of cells or cell beads;

FIG. 3, panels A-F schematically illustrate an example process foramplification and barcoding of cell's nucleic acids;

FIG. 4 provides a schematic illustration of use of barcoding of a cell'snucleic acids in attributing sequence data to individual cells or groupsof cells for use in their characterization;

FIG. 5 provides a schematic illustration of cells associated withlabeled cell-binding ligands;

FIG. 6 shows an example computer control system that is programmed orotherwise configured to implement methods provided herein;

FIG. 7 shows a flowchart for a method of producing droplets containing acell bead and a barcode bead and generating sequence reads frommacromolecular components of the cell bead;

FIG. 8 shows a droplet containing a cell bead and a barcode beadproduced using the method of FIG. 7;

FIG. 9 shows a flowchart for a method of producing droplets containing acell and a barcode bead and generating sequence reads frommacromolecular components of the cell;

FIG. 10 shows a flowchart for a method of producing droplets containinga cell and a barcode bead and generating sequence reads frommacromolecular components of the cell;

FIG. 11 shows a flowchart for a method of producing droplets containinga cross-linked cell and a barcode bead and generating sequence readsfrom macromolecular components the cross-linked cell;

FIG. 12 shows a droplet containing a cross-linked cell and a barcodebead produced using the method of FIG. 11;

FIG. 13 shows a flowchart for a method of producing droplets containinga cell bead and a barcode bead and generating sequence reads frommacromolecular components of the cell bead;

FIG. 14 shows a droplet containing a cell bead and a barcode beadproduced using the method of FIG. 13;

FIG. 15 shows a flowchart for a method of producing droplets containinga cell bead, a barcode bead and generating sequence reads frommacromolecular components of the cell bead;

FIG. 16 shows a droplet containing a cell bead in its own droplet and abarcode bead produced using the method of FIG. 15;

FIG. 17 shows a flowchart for a method of producing droplets containinga coated cell and a barcode bead and generating sequence reads frommacromolecular components the coated cell;

FIG. 18 shows a droplet containing a coated cell and a barcode beadproduced using the method of FIG. 17;

FIG. 19 shows a flowchart for a method of producing droplets containinga cell and barcode bead and generating sequence reads frommacromolecular components of the cell;

FIG. 20 shows a droplet containing a cell and a barcode bead producedusing the method of FIG. 19;

FIG. 21, panels (i)-(v) illustrate an example process of librarypreparation using priming free amplification of templates;

FIG. 22A shows an example method of barcoding amplified templatesgenerated by priming free amplification using an extension barcodingapproach;

FIG. 22B shows an example method of barcoding amplified templatesgenerated by priming free amplification using a single stranded ordouble stranded template to barcode ligation approach;

FIG. 22C shows an example method of barcoding amplified templatesgenerated by the priming free amplification by attaching a single strandDNA molecule (with barcode or primer sequence) to a bead from the 3′end;

FIG. 23 shows a schematic of an example method for retaining longnucleic acid segments and removing short nucleic acid segments;

FIG. 24 shows a schematic of an example method for the amplification andbarcoding of nucleic acid loci from a cell bead;

FIG. 25 shows a flowchart for an example method of producing dropletscontaining cell beads;

FIG. 26A schematically depicts an example droplet comprising a cellbead;

FIG. 26B schematically depicts an example first cell bead comprising asecond cell bead;

FIG. 27 schematically depicts an example method for generating a cellbead in cell bead;

FIGS. 28A and 28B are photographs showing example generation of a cellbead in cell bead;

FIG. 29 depicts example sequencing data obtained from samples preparedin a cell bead in cell bead approach;

FIG. 30 depicts example data depicting centering of a cell in a cellbead in cell bead using different orbital shaking conditions; and

FIG. 31 shows an example of a microfluidic channel structure fordelivering cell beads and barcoded beads to droplets.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information aboutthe analyte. A barcode can be part of an analyte. A barcode can be a tagattached to an analyte (e.g., nucleic acid molecule) or a combination ofthe tag in addition to an endogenous characteristic of the analyte(e.g., size of the analyte or end sequence(s)). A barcode may be unique.Barcodes can have a variety of different formats, for example, barcodescan include: polynucleotide barcodes; random nucleic acid and/or aminoacid sequences; and synthetic nucleic acid and/or amino acid sequences.A barcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads inreal time.

The term “subject,” as used herein, generally refers to an animal, suchas a mammalian species (e.g., human) or avian (e.g., bird) species, orother organism, such as a plant. The subject can be a vertebrate, amammal, a mouse, a primate, a simian or a human. Animals may include,but are not limited to, farm animals, sport animals, and pets. A subjectcan be a healthy or asymptomatic individual, an individual that has oris suspected of having a disease (e.g., cancer) or a pre-disposition tothe disease, or an individual that is in need of therapy or suspected ofneeding therapy. A subject can be a patient.

The term “genome,” as used herein, generally refers to an entirety of asubject's hereditary information. A genome can be encoded either in DNAor in RNA. A genome can comprise coding regions that code for proteinsas well as non-coding regions. A genome can include the sequence of allchromosomes together in an organism. For example, the human genome has atotal of 46 chromosomes. The sequence of all of these together mayconstitute a human genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example,deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), includingvariants or derivatives thereof (e.g., single stranded DNA). Sequencingcan be performed by various systems currently available, such as, withlimitation, a sequencing system by Illumina, Pacific Biosciences, OxfordNanopore, or Life Technologies (Ion Torrent). Such devices may provide aplurality of raw genetic data corresponding to the genetic informationof a subject (e.g., human), as generated by the device from a sampleprovided by the subject. In some situations, systems and methodsprovided herein may be used with proteomic information.

The term “variant,” as used herein, generally refers to a geneticvariant, such as a nucleic acid molecule comprising a polymorphism. Avariant can be a structural variant or copy number variant, which can begenomic variants that are larger than single nucleotide variants orshort indels. A variant can be an alteration or polymorphism in anucleic acid sample or genome of a subject. Single nucleotidepolymorphisms (SNPs) are a form of polymorphisms. Polymorphisms caninclude single nucleotide variations (SNVs), insertions, deletions,repeats, small insertions, small deletions, small repeats, structuralvariant junctions, variable length tandem repeats, and/or flankingsequences. Copy number variants (CNVs), transversions and otherrearrangements are also forms of genetic variation. A genomicalternation may be a base change, insertion, deletion, repeat, copynumber variation, or transversion.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel. Thebead may be formed of a polymeric material. The bead may be magnetic ornon-magnetic.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may be a nucleic acid sampleor protein sample. The biological sample may be derived from anothersample. The sample may be a tissue sample, such as a biopsy, corebiopsy, needle aspirate, or fine needle aspirate. The sample may be afluid sample, such as a blood sample, urine sample, or saliva sample.The sample may be a skin sample. The sample may be a cheek swap. Thesample may be a plasma or serum sample. The sample may be a cell-free orcell free sample. A cell-free sample may include extracellularpolynucleotides. Extracellular polynucleotides may be isolated from abodily sample that may be selected from the group consisting of blood,plasma, serum, urine, saliva, mucosal excretions, sputum, stool andtears.

The term “cell bead,” as used herein, generally refers to a particulatematerial that comprises (e.g., encapsulates, contains, etc.) a cell(e.g., a cell, a fixed cell, a cross-linked cell), a virus, componentsof, or macromolecular constituents derived from a cell or virus. Forexample, a cell bead may comprise a virus and/or a cell. In some cases,a cell bead comprises a single cell. In some cases, a cell bead maycomprise multiple cells adhered together. A cell bead may include anytype of cell, including without limitation prokaryotic cells, eukaryoticcells, bacterial, fungal, plant, mammalian, or other animal cell types,mycoplasmas, normal tissue cells, tumor cells, a T-cell (e.g., CD4T-cell, CD4 T-cell that comprises a dormant copy of humanimmunodeficiency virus (HIV)), a fixed cell, a cross-linked cell, a rarecell from a population of cells, or any other cell type, whether derivedfrom single cell or multicellular organisms. Furthermore, a cell beadmay comprise a live cell, such as, for example, a cell may be capable ofbeing cultured. Moreover, in some examples, a cell bead may comprise aderivative of a cell, such as one or more components of the cell (e.g.,an organelle, a cell protein, a cellular nucleic acid, genomic nucleicacid, messenger ribonucleic acid, a ribosome, a cellular enzyme, etc.).In some examples, a cell bead may comprise material obtained from abiological tissue, such as, for example, obtained from a subject. Insome cases, cells, viruses or macromolecular constituents thereof areencapsulated within a cell bead. Encapsulation can be within a polymeror gel matrix that forms a structural component of the cell bead. Insome cases, a cell bead is generated by fixing a cell in a fixationmedium or by cross-linking elements of the cell, such as the cellmembrane, the cell cytoskeleton, etc. In some cases, beads may or maynot be generated without encapsulation within a larger cell bead.

The term “rare cell,” as used herein, generally refers to a cell whichis present in a sample at a relatively low concentration. The rare cellmay be a cancerous cell. The cancerous cell may be a circulating tumorcell. The rare cell may be obtained from an in vitro fertilization (IVF)procedure. The rare cell may be obtained from an individual displayinggenetic mosaicism. The rare cell may be obtained from an organismproduced using synthetic biology techniques. The rare cell may bepresent at a concentration of at most about 1 in 10², 1 in 10 ³, 1 in10⁴, 1 in 10 ⁵, 1 in 10⁶, 1 in 10⁷, 1 in 10⁸, 1 in 10⁹, 1 in 10 ¹⁰, 1 in10¹¹, 1 in 10¹², 1 in 10¹³, 1 in 10¹⁴, or 1 in 10¹⁵ cells of thepopulation of cells. The rare cell may be present at a concentrationlying in a range defined by any two of the preceding values.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule that is a component of or is derived from abiological material (e.g., a cell, a fixed cell, a cross-linked cell, avirus, etc.). The macromolecular constituent may comprise a nucleicacid. Such a macromolecule can be encapsulated within a cell bead. Themacromolecular constituent may comprise a nucleic acid. Themacromolecular constituent may comprise deoxyribonucleic acid (DNA) or avariant or derivative thereof. The macromolecular constituent maycomprise ribonucleic acid (RNA) or a variant or derivative thereof. TheRNA may be coding or non-coding. The RNA may be messenger RNA (mRNA),ribosomal RNA (rRNA) or transfer RNA (tRNA), for example. The RNA may bea transcript. The RNA may be small RNA that are less than 200 nucleicacid bases in length, or large RNA that are greater than 200 nucleicacid bases in length. Small RNAs may include 5.8 S ribosomal RNA (rRNA),5 S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA(siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA),tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). TheRNA may be double-stranded RNA or single-stranded RNA. The RNA may becircular RNA. The macromolecular constituent may comprise a protein or avariant or derivative thereof. The macromolecular constituent maycomprise a polynucleotide. The macromolecular constituent may comprisemultiple polynucleotides. The macromolecular constituent may compromisechromatin or functional equivalents. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide. The macromolecular constituent may comprise apolynucleotide/polypeptide complex.

The term “tag,” as used herein, generally refers to a material capableof binding to a macromolecular constituent (e.g., DNA, RNA or protein).The tag may bind to the macromolecular constituent with high affinity.The tag may bind to the macromolecular constituent with highspecificity. The tag may comprise a nucleotide sequence. The tag maycomprise an oligonucleotide or polypeptide sequence. The tag maycomprise a DNA aptamer. The tag may be or comprise a primer. The tag maybe or comprise a protein. The tag may comprise a polypeptide. The tagmay be or include a barcode, such as a barcode sequence. The tag may bea molecular species or atomic species (e.g., atomic particle, collectionof atomic particles, or quantum dot).

The term “microfluidic device,” as used herein generally refers to adevice configured for fluid transport and having a fluidic channelthrough which fluid can flow with at least one dimension of no greaterthan about 10 millimeters (mm). The dimension can be any of length,width or height. In some cases, a microfluidic device comprises afluidic channel having multiple dimensions of no greater than about 10mm. A microfluidic device can also include a plurality of fluidicchannels each having a dimension of no greater than about 10 mm. Thedimension(s) of a given fluidic channel of a microfluidic device mayvary depending, for example, on the particular configuration of thechannel and/or channels and other features also included in the device.

In some examples, a dimension of a fluidic channel of a microfluidicdevice may be at most about 10 mm, at most about 9 mm, at most about 8mm, at most about 7 mm, at most about 6 mm, at most about 5 mm, at mostabout 4 mm, at most about 3 mm, at most about 2 mm, at most about 1 mm,at most about 900 micrometers (μm), at most about 800 μm, at most 700μm, at most about 600 μm, at most about 500 μm, at most about 400 μm, atmost about 300 μm, at most about 200 μm, at most about 100 μm, at mostabout 90 μm, at most about 70 μm, at most about 60 μm, at most about 50μm, at most about 40 μm, at most about 30 μm, at most about 20 μm, atmost about 10 μm, at most about 8 μm, at most about 6 μm, at most about4 μm, at most about 2 μm, at most about 1 μm or less. In some examples adimension of a fluidic channel of a microfluidic device may be at leastabout 1 μm, at least about 2 μm, at least about 4 μm, at least about 6μm, at least about 8 μm, at least about 10 μm, at least about 20 μm, atleast about 30 μm, at least about 40 μm, at least about 50 μm, at leastabout 60 μm, at least about 70 μm, at least about 80 μm, at least about90 μm, at least about 100 μm, at least about 200 μm, at least about 300μm, at least about 400 μm, at least about 500 μm, at least about 600 μm,at least about 700 μm, at least about 800 μm, at least about 900 μm, atleast about 1 mm, at least about 2 mm, at least about 3 mm, at leastabout 4 mm, at least about 5 mm, at least about 6 mm, at least about 7mm, at least about 8 mm, at least about 9 mm, at least about 10 mm ormore.

Microfluidic devices described herein can also include any additionalcomponents that can, for example, aid in regulating fluid flow, such asa fluid flow regulator (e.g., a pump, a source of pressure, etc.),features that aid in preventing clogging of fluidic channels (e.g.,funnel features in channels; reservoirs positioned between channels,reservoirs that provide fluids to fluidic channels, etc.) and/orremoving debris from fluid streams, such as, for example, filters.Additional microfluidic features are described in U.S. PatentPublication No. 2015/0292988, which is herein incorporated by referencein its entirety. Moreover, microfluidic devices may be configured as afluidic chip that includes one or more reservoirs that supply fluids toan arrangement of microfluidic channels and also includes one or morereservoirs that receive fluids that have passed through the microfluidicdevice. In addition, microfluidic devices may be constructed of anysuitable material(s), including polymer species and glass.

Nucleic acid sequencing technologies have yielded substantial results insequencing biological materials, including providing substantialsequence information on individual organisms, and relatively purebiological samples. However, these systems have traditionally not beeneffective at being able to identify and characterize cells at the singlecell level.

Many nucleic acid sequencing technologies derive the nucleic acids thatthey sequence from collections of cells obtained from tissue or othersamples, such as biological fluids (e.g., blood, plasma, etc). The cellscan be processed (e.g., all together) to extract the genetic materialthat represents an average of the population of cells, which can then beprocessed into sequencing ready DNA libraries that are configured for agiven sequencing technology. Although often discussed in terms of DNA ornucleic acids, the nucleic acids derived from the cells may include DNA,or RNA, including, e.g., mRNA, total RNA, or the like, that may beprocessed to produce cDNA for sequencing. Following processing, absent acell specific marker, attribution of genetic material as beingcontributed by a subset of cells or an individual cell may not bepossible in such an ensemble approach.

In addition to the inability to attribute characteristics to particularsubsets of cells or individual cells, such ensemble sample preparationmethods can be, from the outset, predisposed to primarily identifyingand characterizing the majority constituents in the sample of cells, andmay not be designed to pick out the minority constituents, e.g., geneticmaterial contributed by one cell, a few cells, or a small percentage oftotal cells in the sample. Likewise, where analyzing expression levels,e.g., of mRNA, an ensemble approach can be predisposed to presentingpotentially inaccurate data from cell populations that arenon-homogeneous in terms of expression levels. In some cases, whereexpression is high in a small minority of the cells in an analyzedpopulation, and absent in the majority of the cells of the population,an ensemble method may indicate low level expression for the entirepopulation.

These inaccuracies can be further magnified through processingoperations used in generating the sequencing libraries from thesesamples. In particular, many next generation sequencing technologies(e.g., massively parallel sequencing) may rely upon the geometricamplification of nucleic acid fragments, such as via polymerase chainreaction, in order to produce sufficient DNA for the sequencing library.However, such amplification can be biased toward amplification ofmajority constituents in a sample, and may not preserve the startingratios of such minority and majority components.

While some of these difficulties may be addressed by utilizing differentsequencing systems, such as single molecule systems that do not requireamplification, the single molecule systems, as well as the ensemblesequencing methods of other next generation sequencing systems, can alsohave large input DNA requirements. Some single molecule sequencingsystems, for example, can have sample input DNA requirements of from 500nanograms (ng) to upwards of 10 micrograms (μg), which may not beobtainable from individual cells or even small subpopulations of cells.Likewise, other NGS systems can be optimized for starting amounts ofsample DNA in the sample of from approximately 50 ng to about 1 μg, forexample.

Disclosed herein are methods and systems for characterizingmacromolecular constituents from small populations of biologicalmaterials (e.g., cells or viruses), and in some cases, forcharacterizing macromolecular constituents from single cells. Themethods described herein may compartmentalize the analysis of individualcells or small populations of cells, including e.g., nucleic acids fromindividual cells or small groups of cells, and then allow that analysisto be attributed back to the individual cell or small group of cellsfrom which the nucleic acids were derived. This can be accomplishedregardless of whether the cell population represents a 50/50 mix of celltypes, a 90/10 mix of cell types, or virtually any ratio of cell types,as well as a complete heterogeneous mix of different cell types, or anymixture between these. Differing cell types may include cells fromdifferent tissue types of an individual or the same tissue type fromdifferent individuals, or biological organisms such as microorganismsfrom differing genera, species, strains, variants, or any combination ofany or all of the foregoing. For example, differing cell types mayinclude normal and tumor tissue from an individual, various cell typesobtained from a human subject such as a variety of immune cells (e.g., Bcells, T cells, and the like), multiple different bacterial species,strains and/or variants from environmental, forensic, microbiome orother samples, or any of a variety of other mixtures of cell types.

In an aspect, the methods and systems described herein, provide for thecompartmentalization, depositing or partitioning of a cell or virus(e.g., a cell) or the macromolecular constituent(s) of the cell or virusfrom a sample into discrete compartments or partitions (referred tointerchangeably herein as partitions), where each partition maintainsseparation of its own contents from the contents of other partitions.These partitions may themselves be partitioned into additionalpartitions, such as, for example, droplets or wells. Unique identifiers,e.g., barcodes, may be previously, subsequently or concurrentlydelivered to the cell or virus or macromolecular constituent(s) of thecell or virus, in order to allow for the later attribution of thecharacteristics of the cell or virus to the particular compartment.Barcodes may be delivered, for example on an oligonucleotide, to apartition via any suitable mechanism.

An overview of an example method 100 for generating partitionscomprising partitions encapsulating a cell (e.g., a fixed cell, across-linked cell) or virus or its macromolecular constituent(s) andbarcodes is schematically depicted in FIG. 1A. Method 100 comprisesthree different phases 110, 120 and 130 that correspond to generation ofcell beads comprising a cell or virus or its macromolecularconstituent(s) (110); solvent exchange to bring generated partitionsinto an aqueous phase, cell or virus lysis and denaturation of the cellor virus or macromolecular constituent(s) of the cell or virus (120);and generation of partitions comprising the generated cell beads andbarcodes and subsequent tagging (e.g., barcoding) (130). With regard tophase 110, an oil 101, polymeric or gel precursors 102 and cells 103 areprovided to a microfluidic chip 104. A photograph of an examplemicrofluidic chip 104 is shown in FIG. 1B. As shown in FIG. 1B, themicrofluidic chip 104 comprises a plurality of reservoirs for the oil101, polymeric or gel precursors 102 and cell or virus reagents 103.Polymeric or gel precursors 102 and cell or virus reagents 103 areflowed (e.g., via the action of an applied force, such as negativepressure via a vacuum or positive pressure via a pump) from theirreservoirs to a first channel junction at which point they combine toform an aqueous stream. This aqueous stream is then flowed to a secondchannel junction, to which oil 101 is also provided. The aqueous streamprovided from the first channel junction is immiscible with the oil 101resulting in the generation of a suspension of aqueous droplets in theoil which then flow to reservoir 105 and represent the product 105 fromthe microfluidic process. Flow can be controlled within the microfluidicchip 104 via any suitable strategy, including the use of one or moreflow regulators in a channel or various channels, dimensioning ofmicrofluidic channels, etc. As shown in both FIG. 1A and FIG. 1B, theproduct comprises droplets 105 comprising a cell from the cells 103 andpolymeric or gel precursors 102.

Continuing with FIG. 1A, the droplets 105 are then subjected toconditions suitable to polymerize or gel the polymeric or gel precursors102 in the droplets 105, which generates cell beads 106 that encapsulatethe cell or virus reagents 103 (e.g., a cell, a fixed cell, across-linked cell, component(s) or a cell) in the droplets 105. As theresulting cell beads 106 are suspended in oil, phase 120 is initiatedwhich includes solvent exchange 111 to resuspend the cell beads 106 inan aqueous phase. Additional details and examples regarding solventexchange are provided elsewhere herein.

The resuspended cell beads 106 can then, in bulk 112, be subjectedconditions suitable to lyse cells or viruses associated with the cellbeads 106 and, separately or contemporaneously, also subjected, in bulk,to conditions to denature nucleic acids derived from the cells orviruses associated with the cell beads 106. The polymeric matrix of thecell beads 106 effectively hinders or prohibits diffusion of largermolecules, such as nucleic acids, from the cell beads 106. The cellbeads 106 are sufficiently porous to denaturation agents that permitdenaturation of trapped nucleic acids within the cell beads 106. In somecases, the cell beads can then be subjected, in bulk, to conditionssuitable for performing one or more reactions on nucleic acids derivedfrom the cells or viruses associated with the cell beads 106. Additionaldetails and examples regarding reactions on nucleic acids are providedelsewhere herein. The resulting cell beads 113 are then collected 114and can be stored prior to initiation of phase 130.

In phase 130, droplets comprising the cell beads 113 and barcode beads(e.g., gel beads) 122 comprising barcode sequences are generated. Asshown in FIG. 1A, an oil 121, the cell beads 113 and barcode beads 122each comprising a barcode sequence (e.g., each bead comprising a uniquebarcode sequence) are provided to a microfluidic chip 123. A photographof an example microfluidic chip 123 is shown in FIG. 1C. As shown inFIG. 1C, the microfluidic chip 123 comprises a plurality of reservoirsfor the oil 121, cell beads 113 and barcode beads 122. The chip alsoincludes additional reservoirs 127 and 128 that may be used to supplyadditional reagents (e.g., reagents for nucleic acid amplification,reagents that can degrade or dissolve cell beads 113 and/or barcodebeads 122, reagents that degrade linkages between barcodes and barcodebeads 122, etc.) to phase 130. Cell beads 113 and barcode beads 122 areflowed (e.g., via the action of an applied force, such as negativepressure via a vacuum or positive pressure via a pump) from theirreservoirs to a first channel junction at which point they combine toform an aqueous mixture. Materials from reservoirs 127 and 128 can alsobe provided to the mixture at the first channel junction.

Alternatively, cell beads and barcode beads can be mixed beforeintroduction into the microfluidic chip. In this case, a singlereservoir of the microfluidic chip 123 comprises a mixture of cell beadsand barcode beads. The ratio of cell beads to barcode beads in themixture can be varied to alter the number of droplets generated thatcomprise a single cell bead and a single barcode bead. The mixture ofcell beads and barcode beads may be flowed (e.g., via the action of anapplied force, such as negative pressure via a vacuum or positivepressure via a pump) from the reservoir to a first channel junction, insome cases together with materials from reservoirs 127 and/or 128. As analternative or in addition to, cells may be mixed with barcode beads.For example, a collection of cells and cell beads may be mixed withbarcode beads, or a collection of cells may be mixed with barcode beads.

In some examples, the mixture comprising cell beads (or cells), barcodebeads, and in some cases additional reagents is then flowed to a secondchannel junction, to which oil 121 is also provided. The aqueous mixtureprovided from the first channel junction is immiscible with the oil 121resulting in the generation of a suspension of aqueous droplets 125 inthe oil 124 which then flow to a reservoir and represent the productfrom the microfluidic process. The microfluidic chip can also include areservoir 129 that can accept excess oil from the stream emerging fromthe second channel. Flow can be controlled within the microfluidic chip123 via any suitable strategy, including the use of one or more flowregulators (see FIGS. 1C and 1D) in a channel or that connect channels,use of various channels, dimensioning of channels, etc. As shown in bothFIG. 1A and FIG. 1C, the product comprises droplets 125 comprising acell bead 113 and a barcode bead 122, in addition to any other reagentsprovided by reservoirs 127 and 128. In some cases, a given droplet ofthe droplets 125 comprises a single cell bead and a single barcode bead.

As in 126 of FIG. 1A, where reagents that degrade or dissolve the cellbeads 113, barcoded beads 122 and/or linkages between barcodes andbarcode beads 122 are present in droplets, these reagents can releasethe nucleic acids trapped in the cell beads 113 from the cell beads 113and release the barcodes from the barcode beads 122. The releasedbarcodes can then interact with the released nucleic acids to generatebarcoded constructs for nucleic acid sequencing as described elsewhereherein. Where a given droplet comprises a single cell bead and a singlebarcode bead comprising oligonucleotides having a common barcodesequence, a given sequencing construct generated from the given droplet125 can be associated with the cell or virus of the given cell bead viaits barcode sequence.

FIG. 1D photographically depicts two example runs demonstrating thegeneration of droplets 125 comprising cell beads and barcode beads usingthe example method shown in FIG. 1A and microfluidic devices depicted inFIGS. 1B and 1C. In FIG. 1D (panel A), droplets comprising cell beadsand barcode beads are shown and in FIG. 1D (panel B) droplets comprisingcell beads comprising magnetic materials and barcode beads are shown.

FIG. 31 shows an example of a microfluidic channel structure 3100 fordelivering barcode carrying beads to droplets. The channel structure3100 can include channel segments 3101, 3102, 3104, 3106 and 3108communicating at a channel junction 3110. In operation, the channelsegment 3101 may transport an aqueous fluid 3112 that includes aplurality of beads 3114 (e.g., with nucleic acid molecules,oligonucleotides, molecular tags) along the channel segment 3101 intojunction 3110. The plurality of beads 3114 may be sourced from asuspension of beads. For example, the channel segment 3101 may beconnected to a reservoir comprising an aqueous suspension of beads 3114.The channel segment 3102 may transport the aqueous fluid 3112 thatincludes a plurality of cell beads 3116 along the channel segment 3102into junction 3110. The plurality of cell beads 3116 may be sourced froma suspension of cell beads. For example, the channel segment 3102 may beconnected to a reservoir comprising an aqueous suspension of cell beads3116. In some instances, the aqueous fluid 3112 in either the firstchannel segment 3101 or the second channel segment 3102, or in bothsegments, can include one or more reagents, as further described below.A second fluid 3118 that is immiscible with the aqueous fluid 3112(e.g., oil) can be delivered to the junction 3110 from each of channelsegments 3104 and 3106. Upon meeting of the aqueous fluid 3112 from eachof channel segments 3101 and 3102 and the second fluid 3118 from each ofchannel segments 3104 and 3106 at the channel junction 3110, the aqueousfluid 3112 can be partitioned as discrete droplets 3120 in the secondfluid 3118 and flow away from the junction 3110 along channel segment3108. The channel segment 3108 may deliver the discrete droplets to anoutlet reservoir fluidly coupled to the channel segment 3108, where theymay be harvested.

As an alternative, the channel segments 3101 and 3102 may meet atanother junction upstream of the junction 3110. At such junction, beadsand cell beads may form a mixture that is directed along another channelto the junction 3110 to yield droplets 3120. The mixture may provide thebeads and cell beads in an alternating fashion, such that, for example,a droplet comprises a single bead and a single cell bead.

Beads, cell beads and droplets may flow along channels at substantiallyregular flow profiles (e.g., at regular flow rates). Such regular flowprofiles may permit a droplet to include a single bead and a single cellbead. Such regular flow profiles may permit the droplets to have anoccupancy (e.g., droplets having beads and cell beads) greater than 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flowprofiles and devices that may be used to provide such regular flowprofiles are provided in, for example, U.S. Patent Publication No.2015/0292988, which is entirely incorporated herein by reference.

The second fluid 3118 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets3120.

A discrete droplet that is generated may include an individual cell bead3116. A discrete droplet that is generated may include a barcode orother reagent carrying bead 3114. A discrete droplet generated mayinclude both an individual cell bead and a barcode carrying bead, suchas droplets 3120. In some instances, a discrete droplet may include morethan one individual cell bead or no cell bead. In some instances, adiscrete droplet may include more than one bead or no bead. A discretedroplet may be unoccupied (e.g., no beads, no cell beads).

Beneficially, a discrete droplet partitioning a cell bead and a barcodecarrying bead may effectively allow the attribution of the barcode tomacromolecular constituents of the cell bead within the partition. Thecontents of a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 3100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A partition may be a droplet. The droplet may be formed by bringing afirst phase in contact with a second phase that is immiscible with thefirst phase. As an alternative, the partition may be a well as part of aplurality of wells. As another alternative, the partition may be achamber as part of a plurality of chambers. Partitions may befluidically isolated from one another.

In some embodiments, barcoded oligonucleotides are delivered to apartition via a microcapsule, such as a bead (e.g., gel bead) or adroplet. In some cases, barcoded oligonucleotides are initiallyassociated with the microcapsule and then released from the microcapsuleupon application of a stimulus which allows the oligonucleotides todissociate or to be released from the microcapsule.

A microcapsule, in some embodiments, comprises a bead, such as a dropletcomprising the bead. As an alternative, the microcapsule can be a bead(e.g., gel bead). In some embodiments, a bead may be porous, non-porous,solid, semi-solid, semi-fluidic, or fluidic. In some embodiments, a beadmay be dissolvable, disruptable, or degradable. In some cases, a beadmay not be degradable. The bead may be a solid or semi-solid particle.In some embodiments, the bead may be a gel bead. A gel bead may be ahydrogel bead. A gel bead may be formed from molecular precursors, suchas a polymeric or monomeric species. A semi-solid bead may be aliposomal bead. Solid beads may comprise metals including iron oxide,gold, and silver. In some cases, the beads are silica beads. In somecases, the beads are rigid. In some cases, the beads may be flexibleand/or compressible.

In some embodiments, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the precursors. In some cases, a precursor may be analready polymerized species capable of undergoing further polymerizationvia, for example, a chemical cross-linkage. In some cases, a precursorcomprises one or more of an acrylamide or a methacrylamide monomer,oligomer, or polymer. In some cases, the bead may comprise prepolymers,which are oligomers capable of further polymerization. For example,polyurethane beads may be prepared using prepolymers. In some cases, thebead may contain individual polymers that may be further polymerizedtogether. In some cases, beads may be generated via polymerization ofdifferent precursors, such that they comprise mixed polymers,co-polymers, and/or block co-polymers.

A bead may comprise natural and/or synthetic materials. For example, apolymer can be a natural polymer or a synthetic polymer. In some cases,a bead comprises both natural and synthetic polymers. Examples ofnatural polymers include proteins and sugars such as deoxyribonucleicacid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins,enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan,dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin,shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gumkaraya, agarose, alginic acid, alginate, or natural polymers thereof.Examples of synthetic polymers include acrylics, nylons, silicones,spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate,polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes,polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene,polycarbonate, polyethylene, polyethylene terephthalate,poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethyleneterephthalate), polyethylene, polyisobutylene, poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde, polypropylene,polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene dichloride),poly(vinylidene difluoride), poly(vinyl fluoride) and combinations(e.g., co-polymers) thereof. Beads may also be formed from materialsother than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some cases, a chemical cross-linker may be a precursor used tocross-link monomers during polymerization of the monomers and/or may beused to attach oligonucleotides (e.g., barcoded oligonucleotides) to thebead. In some cases, polymers may be further polymerized with across-linker species or other type of monomer to generate a furtherpolymeric network. Non-limiting examples of chemical cross-linkers (alsoreferred to as a “crosslinker” or a “crosslinker agent” herein) includecystamine, gluteraldehyde, dimethyl suberimidate, N-Hydroxysuccinimidecrosslinker BS3, formaldehyde, carbodiimide (EDC), SMCC, Sulfo-SMCC,vinylsilane, N,N′diallyltartardiamide (DATD),N,N′-Bis(acryloyl)cystamine (BAC), or homologs thereof. In some cases,the crosslinker used in the present disclosure contains cystamine.

Crosslinking may be permanent or reversible, depending upon theparticular crosslinker used. Reversible crosslinking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some embodiments, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and oligonucleotides. Cystamine(including modified cystamines), for example, is an organic agentcomprising a disulfide bond that may be used as a crosslinker agentbetween individual monomeric or polymeric precursors of a bead.Polyacrylamide may be polymerized in the presence of cystamine or aspecies comprising cystamine (e.g., a modified cystamine) to generatepolyacrylamide gel beads comprising disulfide linkages (e.g., chemicallydegradable beads comprising chemically-reducible cross-linkers). Thedisulfide linkages may permit the bead to be degraded (or dissolved)upon exposure of the bead to a reducing agent.

In some embodiments, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some embodiments, the bead may comprise covalent or ionic bondsbetween polymeric precursors (e.g., monomers, oligomers, linearpolymers), oligonucleotides, primers, and other entities. In some cases,the covalent bonds comprise carbon-carbon bonds or thioether bonds.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more oligonucleotides (e.g.,barcode sequence, barcoded oligonucleotide, primer, or otheroligonucleotide) to the bead. In some cases, an acrydite moiety canrefer to an acrydite analogue generated from the reaction of acryditewith one or more species, such as, the reaction of acrydite with othermonomers and cross-linkers during a polymerization reaction. Acryditemoieties may be modified to form chemical bonds with a species to beattached, such as an oligonucleotide (e.g., barcode sequence, barcodedoligonucleotide, primer, or other oligonucleotide). Acrydite moietiesmay be modified with thiol groups capable of forming a disulfide bond ormay be modified with groups already comprising a disulfide bond. Thethiol or disulfide (via disulfide exchange) may be used as an anchorpoint for a species to be attached or another part of the acryditemoiety may be used for attachment. In some cases, attachment isreversible, such that when the disulfide bond is broken (e.g., in thepresence of a reducing agent), the attached species is released from thebead. In other cases, an acrydite moiety comprises a reactive hydroxylgroup that may be used for attachment.

Functionalization of beads for attachment of oligonucleotides may beachieved through a wide range of different approaches, includingactivation of chemical groups within a polymer, incorporation of activeor activatable functional groups in the polymer structure, or attachmentat the pre-polymer or monomer stage in bead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to an oligonucleotide, such as aprimer (e.g., a primer for amplifying target nucleic acids, barcodedoligonucleotide, etc) to be incorporated into the bead. In some cases,the primer comprises a P5 sequence for attachment to a sequencing flowcell for Illumina sequencing. In some cases, the primer comprises a P7sequence for attachment to a sequencing flow cell for Illuminasequencing. In some cases, the primer comprises a barcode sequence. Insome cases, the primer further comprises a unique molecular identifier(UMI). In some cases, the primer comprises an R1 primer sequence forIllumina sequencing. In some cases, the primer comprises an R2 primersequence for Illumina sequencing.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat a small number of disulfide linkages are activated. Control may beexerted, for example, by controlling the concentration of a reducingagent used to generate free thiol groups and/or concentration ofreagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than about 10,000, less than about 100,000, less thanabout 1,000,000, less than about 10,000,000, less than about100,000,000, less than about 1,000,000,000, less than about10,000,000,000, or less than about 100,000,000,000) of reducing agentmay be used for reduction. Controlling the number of disulfide linkagesthat are reduced to free thiols may be useful in ensuring beadstructural integrity during functionalization. In some cases,optically-active agents, such as fluorescent dyes may be may be coupledto beads via free thiol groups of the beads and used to quantify thenumber of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature or temperaturechange, subjecting the beads to a higher or lower ion concentration,and/or subjecting the beads to an electric field. The de-swelling of thebeads may be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to a loweror high temperature or temperature change different from that use toswell the beads, subjecting the beads to a lower or higher ionconcentration different from that used to swell the beads, and/orremoving the electric field.

Transferring the beads may cause pores in the beads to shrink. Suchshrinking may then hinder reagents within the beads from diffusing outof the interiors of the beads. The hindrance may be due to stericinteractions between the reagents and the interiors of the beads. Thetransfer may be accomplished microfluidically. For instance, thetransfer may be achieved by moving the beads from one co-flowing solventstream to a different co-flowing solvent stream. The swellability and/orpore size of the beads may be adjusted by changing the polymercomposition of the bead.

In some cases, an acrydite moiety linked to precursor, another specieslinked to a precursor, or a precursor itself comprises a labile bond,such as chemically, thermally, or photo-sensitive bonds e.g., disulfidebonds, UV sensitive bonds, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, etc.) such that release ofspecies attached to a bead via each labile bond may be controlled by theapplication of the appropriate stimulus. Such functionality may beuseful in controlled release of species from a gel bead. In some cases,another species comprising a labile bond may be linked to a gel beadafter gel bead formation via, for example, an activated functional groupof the gel bead as described above. Barcodes that are releasably,cleavably or reversibly attached to the beads described herein includebarcodes that are released or releasable through cleavage of a linkagebetween the barcode molecule and the bead, or that are released throughdegradation of the underlying bead itself, allowing the barcodes to beaccessed or accessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)).

Species that do not participate in polymerization may also beencapsulated in beads during bead generation (e.g., duringpolymerization of precursors). Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,oligonucleotides, reagents for a nucleic acid amplification reaction(e.g., primers (e.g. random primers, primers specific for a given DNAloci), polymerases, nucleotides (e.g. unmodified nucleotides, modifiednucleotides, or non-canonical nucleotides), co-factors (e.g., ionicco-factors)) including those described herein, reagents for enzymaticreactions (e.g., enzymes, co-factors, substrates), reagents for reversetranscription (e.g. oligonucleotide primers or reverse transcriptase),or reagents for nucleic acid modification reactions such aspolymerization, ligation, digestion, methylation, random mutagenesis,bisulfite conversion, uracil hydrolysis, nucleic acid repair, nucleicacid insertion or cleavage (e.g. via CRISPR/Cas9-mediated ortransposon-mediated insertion or cleavage), capping, or decapping.Trapping of such species may be controlled by the polymer networkdensity generated during polymerization of precursors, control of ioniccharge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. In some cases, barcode sequences (e.g., oligonucleotidescomprising barcode sequences) may also be encapsulated within a beadand, in some cases, can be released from a bead via bead degradationand/or by application of a stimulus capable of releasing the speciesfrom the bead.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm,50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm. Insome cases, a bead may have a diameter of at least about 1 μm, 5 μm, 10μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250μm, 500 μm, 1 mm, or more. In some cases, a bead may have a diameter ofless than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm. In some cases, a beadmay have a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm,40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500μm.

In certain aspects, beads are provided as a population or plurality ofbeads having a relatively monodisperse size distribution. To providerelatively consistent amounts of reagents within partitions, maintainingrelatively consistent bead characteristics, such as size, can contributeto the overall consistency. In particular, the beads described hereinmay have size distributions that have a coefficient of variation intheir cross-sectional dimensions of less than 50%, less than 40%, lessthan 30%, less than 20%, and in some cases less than 15%, less than 10%,or less than 5%.

Beads may be of any suitable shape. Examples of bead shapes include, butare not limited to, spherical, non-spherical, oval, oblong, amorphous,circular, cylindrical, and variations thereof.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, e.g., barcode containingoligonucleotides, described above, the beads may be degradable,disruptable, or dissolvable spontaneously or upon exposure to one ormore stimuli (e.g., temperature changes, pH changes, exposure toparticular chemical species or phase, exposure to light, reducing agent,etc.). In some cases, a bead may be dissolvable, such that materialcomponents of the beads are solubilized when exposed to a particularchemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead is degraded ordissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a oligonucleotide, e.g., barcoded oligonucleotide) may result in releaseof the species from the bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., an oligonucleotide, a barcode sequence, a primer, etc)from the bead when the appropriate stimulus is applied to the bead ascompared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides) may interact with otherreagents contained in the partition. For example, a polyacrylamide beadcomprising cystamine and linked, via a disulfide bond, to a barcodesequence, may be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent breaks thevarious disulfide bonds resulting in bead degradation and release of thebarcode sequence into the aqueous, inner environment of the droplet. Inanother example, heating of a droplet comprising a bead-bound barcodesequence in basic solution may also result in bead degradation andrelease of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

While referred to as degradation of a bead, in many instances as notedabove, that degradation may refer to the disassociation of a bound orentrained species from a bead, both with and without structurallydegrading the physical bead itself. For example, entrained species maybe released from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

Where degradable beads are provided, it may helpful to avoid exposingsuch beads to the stimulus or stimuli that cause such degradation priorto the requisite time, in order to avoid premature bead degradation andissues that arise from such degradation, including for example poor flowcharacteristics and aggregation. By way of example, where beads comprisereducible cross-linking groups, such as disulfide groups, it can behelpful to avoid contacting such beads with reducing agents, e.g., DTTor other disulfide cleaving reagents. In such cases, treatment to thebeads described herein will, in some cases be provided free of reducingagents, such as DTT. Because reducing agents are often provided incommercial enzyme preparations, it may be helpful to provide reducingagent free (or DTT free) enzyme preparations in treating the beadsdescribed herein. Examples of such enzymes include, e.g., polymeraseenzyme preparations, reverse transcriptase enzyme preparations, ligaseenzyme preparations, as well as many other enzyme preparations that maybe used to treat the beads described herein. The terms “reducing agentfree” or “DTT free” preparations can refer to a preparation having lessthan 1/10th, less than 1/50th, and even less than 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation will typically have less than0.01 mM, 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less than 0.0001mM DTT. In many cases, the amount of DTT will be undetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

The methods, compositions, devices, and kits of this disclosure may beused with any suitable agent to degrade beads. In some embodiments,changes in temperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM. The reducing agent may be presentat a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM,or greater than 10 mM. The reducing agent may be present at aconcentration of at most about 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM.

Any suitable number of barcode molecules (e.g., primer, e.g., barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the barcode molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer is limited by the process of producingoligonucleotide bearing beads.

The compartments or partitions can comprise partitions that are flowablewithin fluid streams. These partitions may comprise, e.g.,micro-vesicles that have an outer barrier surrounding an inner fluidcenter or core, or, in some cases, they may comprise a porous matrixthat is capable of entraining and/or retaining materials within itsmatrix. Partitions can comprise droplets of aqueous fluid within anon-aqueous continuous phase, e.g., an oil phase. A variety of differentvessels are described in, for example, U.S. Patent ApplicationPublication No. 2014/0155295, which is entirely incorporated herein byreference for all purposes. Emulsion systems for creating stabledroplets in non-aqueous or oil continuous phases are described in detailin, e.g., U.S. Patent Application Publication No. 2010/0105112, which isentirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual cell beadsto discrete partitions may generally be accomplished by introducing aflowing stream of cell beads in an aqueous fluid into a flowing streamof a non-aqueous fluid, such that droplets are generated at the junctionof the two streams. By providing the aqueous stream at a certainconcentration of cell beads, the occupancy of the resulting partitions(e.g., number of cell beads per partition) can be controlled. Wheresingle cell bead partitions are implemented, the relative flow rates ofthe fluids can be selected such that, on average, the partitions containless than one cell bead per partition, in order to ensure that thosepartitions that are occupied, are primarily singly occupied. In someembodiments, the relative flow rates of the fluids can be selected suchthat a majority of partitions are occupied, e.g., allowing for a smallpercentage of unoccupied partitions. The flows and channel architecturescan be controlled as to ensure a requisite number of singly occupiedpartitions, less than a certain level of unoccupied partitions and lessthan a certain level of multiply occupied partitions.

The systems and methods described herein can be operated such that amajority of occupied partitions include no more than one cell bead peroccupied partition. In some cases, the partitioning process is conductedsuch that fewer than 40% of the occupied partitions contain more thanone cell bead, fewer than 35% of the occupied partitions contain morethan one cell bead, fewer than 30% of the occupied partitions containmore than one cell bead, fewer than 25% of the occupied partitionscontain more than one cell bead, fewer than 20% of the occupiedpartitions contain more than one cell bead, fewer than 15% of theoccupied partitions contain more than one cell bead, fewer than 10% ofthe occupied partitions contain more than one cell bead, or fewer than5% of the occupied partitions include more than one cell bead perpartition.

In some cases, it can be helpful to avoid the creation of excessivenumbers of empty partitions or partitions that do not include a cellbead. For example, from a cost perspective and/or efficiencyperspective, it may helpful to minimize the number of empty partitions.While this may be accomplished by providing sufficient numbers of cellbeads into the partitioning zone, the Poissonian distribution mayexpectedly increase the number of partitions that may include multiplecell beads. As such, in accordance with aspects described herein, theflow of one or more of the cell beads, or other fluids directed into apartitioning zone can be manipulated to control occupancy of partitionswith cell beads such that no more than 60% of the generated partitionsare unoccupied, no more than 50% of the generated partitions areunoccupied, no more than 45% of the generated partitions are unoccupied,no more than 40% of the generated partitions are unoccupied, no morethan 35% of the generated partitions are unoccupied, no more than 30% ofthe generated partitions are unoccupied, no more than 25% of thegenerated partitions are unoccupied, no more than 20% of the generatedpartitions are unoccupied, or no more than 10% of the generatedpartitions are unoccupied. These flows can be controlled so as topresent non-Poissonian distribution of single occupied partitions whileproviding lower levels of unoccupied partitions.

The above noted ranges of unoccupied partitions can be achieved whilestill providing any of the single occupancy rates described above. Forexample, in many cases, the use of the systems and methods describedherein creates resulting partitions (e.g., droplets comprising cellbeads) that have multiple occupancy rates of less than 50%, less than45%, less than 40%, less than 35%, less than 30%, less than 25%, lessthan 20%, less than 15%, less than 10%, less than 5% or less than 1%.

The above-described occupancy rates are also applicable to partitionsthat include both cell beads and additional reagents, including, but notlimited to, microcapsules or particles (e.g., beads, gel beads) carryingbarcoded oligonucleotides. The occupied partitions (e.g., at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupiedpartitions) can include both a microcapsule (e.g., bead) comprisingbarcoded oligonucleotides and a cell bead.

Although described in terms of providing substantially singly occupiedpartitions, above, in certain cases, it is helpful to provide multiplyoccupied partitions, e.g., containing two, three, four or more cellbeads and/or microcapsules (e.g., beads, gel beads) comprising barcodedoligonucleotides within a single partition. Accordingly, as noted above,the flow characteristics of the cell bead and/or bead containing fluidsand partitioning fluids may be controlled to provide for such multiplyoccupied partitions. In particular, the flow parameters may becontrolled to provide a requisite occupancy rate at greater than 50% ofthe partitions, greater than 55% of the partitions, greater than 60% ofthe partitions, greater than 65% of the partitions, greater than 70% ofthe partitions, greater than 75% of the partitions, greater than 80% ofthe partitions, greater than 85% of the partitions, greater than 90% ofthe partitions, greater than 95% of the partitions, or higher.

In some cases, additional microcapsules are used to deliver additionalreagents to a partition. In such cases, it may be advantageous tointroduce different beads into a common channel or droplet generationjunction, from different bead sources, i.e., containing differentassociated reagents, through different channel inlets into such commonchannel or droplet generation junction. In such cases, the flow andfrequency of the different beads into the channel or junction may becontrolled to provide for the requisite ratio of microcapsules from eachsource, while ensuring the requisite pairing or combination of suchbeads into a partition with the requisite number of cell beads.

The partitions described herein may comprise small volumes, e.g., lessthan 10 μL, less than 5 μL, less than 1 μL, less than 900 picoliters(pL), less than 800 pL, less than 700 pL, less than 600 pL, less than500 pL, less than 400 pL, less than 300 pL, less than 200 pL, less than100 pL, less than 50 pL, less than 20 pL, less than 10 pL, less than 1pL, less than 500 nanoliters (nL), or even less than 100 nL, 50 nL, oreven less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than 1000 pL, less than 900 pL, lessthan 800 pL, less than 700 pL, less than 600 pL, less than 500 pL, lessthan 400 pL, less than 300 pL, less than 200 pL, less than 100 pL, lessthan 50 pL, less than 20 pL, less than 10 pL, or even less than 1 pL.Where co-partitioned with microcapsules, the sample fluid volume, e.g.,including co-partitioned cell beads, within the partitions may be lessthan 90% of the above described volumes, less than 80%, less than 70%,less than 60%, less than 50%, less than 40%, less than 30%, less than20%, or even less than 10% the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated to generate the plurality ofpartitions. For example, in a method described herein, a plurality ofpartitions may be generated that comprises at least about 1,000partitions, at least about 5,000 partitions, at least about 10,000partitions, at least about 50,000 partitions, at least about 100,000partitions, at least about 500,000 partitions, at least about 1,000,000partitions, at least about 5,000,000 partitions at least about10,000,000 partitions, at least about 50,000,000 partitions, at leastabout 100,000,000 partitions, at least about 500,000,000 partitions orat least about 1,000,000,000 partitions. Moreover, the plurality ofpartitions may comprise both unoccupied partitions (e.g., emptypartitions) and occupied partitions.

FIG. 2 shows an example of a simplified microfluidic channel structurefor partitioning individual cell beads (e.g., a fixed cell, across-linked cell, a polymer particle comprising a cell). As describedelsewhere herein, in some cases, the majority of occupied partitionsinclude no more than one cell bead per occupied partition and, in somecases, some of the generated partitions are unoccupied. In some cases,though, some of the occupied partitions may include more than one cellbead. In some cases, the partitioning process may be controlled suchthat fewer than 25% of the occupied partitions contain more than onecell bead, fewer than 20% of the occupied partitions have more than onecell bead, while in some cases, fewer than 10% or even fewer than 5% ofthe occupied partitions include more than one cell bead per partition.As shown, the channel structure can include channel segments 232, 234,236 and 238 communicating at a channel junction 240. In operation, afirst aqueous fluid 242 that includes suspended cell bead 244, may betransported along channel segment 232 into junction 240, while a secondfluid 246 that is immiscible with the aqueous fluid 242 is delivered tothe junction 240 from channel segments 234 and 236 to create discretedroplets 118 of the aqueous fluid including individual cell bead 244,flowing into channel segment 238.

This second fluid 246 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,e.g., inhibiting subsequent coalescence of the resulting droplets.Examples of particularly useful partitioning fluids andfluorosurfactants are described for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, a cell, virus, components thereof, or macromolecularconstituents thereof may be encapsulated within a cell bead.Encapsulation of a cell, virus, components thereof, or macromolecularconstituents thereof may be performed by a variety of processes. Suchprocesses combine an aqueous fluid containing the a cell, virus,components thereof, or macromolecular constituents thereof to beanalyzed with a polymeric precursor material that may be capable ofbeing formed into a gel or other solid or semi-solid matrix uponapplication of a particular stimulus to the polymer precursor. Suchstimuli include, e.g., thermal stimuli (either heating or cooling),photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g.,through crosslinking, polymerization initiation of the precursor (e.g.,through added initiators), or the like.

Preparation of cell beads comprising a cell, virus, components thereof,or macromolecular constituents thereof may be performed by a variety ofmethods. For example, air knife droplet or aerosol generators may beused to dispense droplets of precursor fluids into gelling solutions inorder to form cell beads that include individual a cell, virus,components thereof, or macromolecular constituents thereof. Likewise,membrane based encapsulation systems may be used to generate cell beadscomprising encapsulated a cell, virus, components thereof, ormacromolecular constituents thereof as described herein. Microfluidicsystems of the present disclosure, such as that shown in FIG. 2, may bereadily used in encapsulating cells as described herein. In particular,and with reference to FIG. 2, the aqueous fluid comprising the cells andthe polymer precursor material is flowed into channel junction 240,where it is partitioned into droplets 248 comprising the individualcells 244, through the flow of non-aqueous fluid 246. In the case ofencapsulation methods, non-aqueous fluid 246 may also include aninitiator to cause polymerization and/or crosslinking of the polymerprecursor to form the microcapsule that includes the entrained cells.Examples of polymer precursor/initiator pairs include those described inU.S. Patent Application Publication No. 2014/0378345, which is entirelyincorporated herein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, e.g., a linear polyacrylamide, PEG, or otherlinear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams in channel segments 234 and 236, which initiates thecopolymerization of the acrylamide and BAC into a cross-linked polymernetwork or, hydrogel.

Upon contact of the second fluid stream 246 with the first fluid stream242 at junction 240 in the formation of droplets, the TEMED may diffusefrom the second fluid 246 into the aqueous first fluid 242 comprisingthe linear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets, resulting in the formation of thegel, e.g., hydrogel, microcapsules 248, as solid or semi-solid beads orparticles entraining the cells 244. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions, e.g., Ca2+, can beused as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling, e.g., upon cooling, or the like.

In some cases, an encapsulated cell, virus, components thereof, ormacromolecular constituents thereof can be selectively releasable fromthe microcapsule, e.g., through passage of time, or upon application ofa particular stimulus, that degrades the microcapsule sufficiently toallow the cell, or its contents to be released from the microcapsule,e.g., into a partition, such as a droplet. For example, in the case ofthe polyacrylamide polymer described above, degradation of themicrocapsule may be accomplished through the introduction of anappropriate reducing agent, such as DTT or the like, to cleave disulfidebonds that cross link the polymer matrix (see, e.g., U.S. PatentApplication Publication No. 2014/0378345, which is entirely incorporatedherein by reference for all purposes).

In accordance with certain aspects, the cell beads may be contacted withlysis reagents in order to release the contents of cells or virusesassociated with the cell bead. In some cases, the lysis agents can becontacted with a cell bead suspension in bulk after cell bead formation.Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Mo.), a surfactant based lysis solution(e.g., TRITON X-100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethyleneglycol), Tween 20, sodium dodecyl sulfate (SDS)) for example, as well asother commercially available lysis enzymes. Electroporation, thermal,acoustic or mechanical cellular disruption may also be used in certaincases. In some cases, such methods give rise to a pore size that issufficiently small to retain nucleic acid fragments of a particularsize, following cellular disruption.

Other reagents can also be contacted with the cell beads, including, forexample, DNase and RNase inactivating agents or inhibitors, such asproteinase K, chelating agents, such as EDTA, and other reagentsemployed in removing or otherwise reducing negative activity or impactof different cell lysate components on subsequent processing of nucleicacids. In addition, in the case of encapsulated cell beads, the cellbeads may be exposed to an appropriate stimulus to release the cellbeads or their contents from a co-partitioned microcapsule. For example,in some cases, a chemical stimulus may be co-partitioned along with anencapsulated cell bead to allow for the degradation of the microcapsuleand release of the cell or its contents into the larger partition. Insome cases, this stimulus may be the same as the stimulus describedelsewhere herein for release of oligonucleotides from their respectivemicrocapsule (e.g., bead). In alternative aspects, this may be adifferent and non-overlapping stimulus, in order to allow anencapsulated cell bead release its contents into a partition at adifferent time from the release of oligonucleotides into the samepartition.

Additional reagents may also be co-partitioned with the cell beads. Insome instances, reagents may be encapsulated within the cell beads. Inother instances, reagents may be outside the cell beads. Reagents may bethose useful in modification of a cell bead's nucleic acid (e.g., DNA,RNA, etc.), where such modification may include ligation, digestion,methylation, random mutagenesis, bisulfite conversion, uracilhydrolysis, nucleic acid repair, capping, or decapping. Additionalreagents may also include reagents useful in amplification of a cellbead's nucleic acid, including primers (e.g. random primers, primersspecific for given DNA loci), polymerases, nucleotides (e.g. unmodifiednucleotides, modified nucleotides, or non-canonical nucleotides), orco-factors (e.g., ionic co-factors). Additional reagents may alsoinclude proteases to remove proteins bound to a cell bead's nucleicacids and transposons to fragment or insert a known sequence into a cellbead's DNA. Additional reagents may also include a nucleic acid, a Cas9nuclease and a guide RNA to mediate editing of a cell bead's DNA.Additional reagents may also include endonucleases to fragment a cellbead's DNA, DNA polymerase enzymes and nucleotides used to amplify thecell bead's nucleic acid fragments and to attach the barcodes to theamplified fragments. Additional reagents may also include reversetranscriptase enzymes, including enzymes with terminal transferaseactivity, primers and oligonucleotides, and switch oligonucleotides(also referred to herein as “switch oligos” or “template switchingoligonucleotides”) which can be used for template switching. In somecases, template switching can be used to increase the length of a cDNA.In some cases, template switching can be used to append a predefinednucleic acid sequence to the cDNA. In an example of template switching,cDNA can be generated from reverse transcription of a template, e.g.,cellular mRNA, where a reverse transcriptase with terminal transferaseactivity can add additional nucleotides, e.g., polyC, to the cDNA in atemplate independent manner. Switch oligos can include sequencescomplementary to the additional nucleotides, e.g., polyG. The additionalnucleotides (e.g., polyC) on the cDNA can hybridize to the additionalnucleotides (e.g., polyG) on the switch oligo, whereby the switch oligocan be used by the reverse transcriptase as template to further extendthe cDNA. Template switching oligonucleotides may comprise ahybridization region and a template region. The hybridization region cancomprise any sequence capable of hybridizing to the target. In somecases, as previously described, the hybridization region comprises aseries of G bases to complement the overhanging C bases at the 3′ end ofa cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases,3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

Macromolecular components may be processed (e.g., subjected to nucleicacid amplification) prior to generation of cell beads. Alternatively orin addition, macromolecular components contained within the cell beadsmay be further processed. Further processing may, in some instances,occur prior to partitioning of the cell beads into discrete partitions.Further processing may also occur following partitioning of the cellbeads into discrete partitions and prior to release of the contents ofthe cell beads into their respective partitions. Alternatively oradditionally, further processing may occur once the contents of the cellbeads are released into their respective partitions. Further processingmay include, for example, nucleic acid modification, where suchmodification may include ligation, digestion, methylation, randommutagenesis, bisulfite conversion, uracil hydrolysis, nucleic acidrepair, capping, or decapping. Further processing may also includenucleic acid amplification, including isothermal amplification (e.g.,loop mediated isothermal amplification or multiple displacementamplification) or PCR (e.g., DOP-PCR), where amplification mayincorporate unmodified bases, modified bases, or non-canonical bases.Additional processing may also include nucleic acid insertion orcleavage (e.g., via CRISPR/Cas9-mediated or transposon-mediatedinsertion or cleavage). Additional processing may also include reversetranscription, where reverse transcription may incorporate unmodifiedbases, modified bases, or non-canonical bases.

Nucleic acid amplification may include performing one or more extensionreactions. Such one or more extension reactions may be performed using aprimer or multiple primers. Nucleic acid amplification may generate oneor more copies of a starting molecule. In some examples, nucleic acidamplification includes a single extension reaction without anyadditional extension reactions. In such a case, for example, nucleicacid amplification may generate a larger molecule from a smallerstarting molecule without generating a copy of the smaller startingmolecule or the larger molecule. However, in some cases, nucleic acidamplification may include generating the larger molecule andsubsequently generating one or more copies of the larger molecule.Nucleic acid amplification may be exponential amplification.Alternatively, nucleic acid amplification may not be exponentialamplification (e.g., may be linear amplification).

Examples of nucleic acid amplification are provided elsewhere herein.Nucleic acid amplification may be isothermal amplification, PCR (e.g.,DOP-PCR) or PHASE, for example. In some cases, nucleic acidamplification may not be PCR.

In some cases, a cell bead comprising a nucleic acid molecule may beprovided in a partition (e.g., droplet), the nucleic acid molecule maybe released from the cell bead in the partition, and the nucleic acidmolecule may be recovered from the partition without any processing. Thenucleic acid molecule may then be processed once recovered from thepartition. For example, the nucleic acid molecule may be subjected tonucleic acid amplification and/or sequencing.

In accordance with the methods and systems described herein, themacromolecular component contents of individual cell beads can beprovided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same cell bead or particles (and, thus, cell or virusoriginally associated with the cell bead). The ability to attributecharacteristics to a cell, virus, components thereof, or macromolecularconstituents thereof of individual cell beads or groups of cell beads isprovided by the assignment of unique identifiers specifically to anindividual cell bead or groups of cell beads. Unique identifiers, e.g.,in the form of nucleic acid barcodes can be assigned or associated withindividual cell beads or populations of cell bead, in order to tag orlabel the cell bead's macromolecular components (and as a result, itscharacteristics) with the unique identifiers. These unique identifierscan then be used to attribute the cell bead's components andcharacteristics to the original cell or virus(s) associated with thecell bead. In some aspects, this is performed by co-partitioning theindividual cell bead or groups of cell beads with the uniqueidentifiers. In some aspects, the unique identifiers are provided in theform of oligonucleotides that comprise nucleic acid barcode sequencesthat may be attached to or otherwise associated with the nucleic acidcontents of individual cell bead, or to other components of the cellbead, and particularly to fragments of those nucleic acids. Theoligonucleotides are partitioned such that as between oligonucleotidesin a given partition, the nucleic acid barcode sequences containedtherein are the same, but as between different partitions, theoligonucleotides can, and do have differing barcode sequences, or atleast represent a large number of different barcode sequences across allof the partitions in a given analysis. In some aspects, only one nucleicacid barcode sequence can be associated with a given partition, althoughin some cases, two or more different barcode sequences may be present.

The nucleic acid barcode sequences can include from 6 to about 20 ormore nucleotides within the sequence of the oligonucleotides. In somecases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides maybe completely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may be 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned oligonucleotides can also comprise other functionalsequences useful in the processing of the nucleic acids from theco-partitioned cell beads. These sequences include, e.g., targeted orrandom/universal amplification primer sequences for amplifying thegenomic DNA from the individual cell beads within the partitions whileattaching the associated barcode sequences, sequencing primers or primerrecognition sites, hybridization or probing sequences, e.g., foridentification of presence of the sequences or for pulling down barcodednucleic acids, or any of a number of other potential functionalsequences. Other mechanisms of co-partitioning oligonucleotides may alsobe employed, including, e.g., coalescence of two or more droplets, whereone droplet contains oligonucleotides, or microdispensing ofoligonucleotides into partitions, e.g., droplets within microfluidicsystems.

In an example, microcapsules, such as beads, are provided that eachincludes large numbers of the above described barcoded oligonucleotidesreleasably attached to the beads, where all of the oligonucleotidesattached to a particular bead will include the same nucleic acid barcodesequence, but where a large number of diverse barcode sequences arerepresented across the population of beads used. In some embodiments,hydrogel beads, e.g., comprising polyacrylamide polymer matrices, areused as a solid support and delivery vehicle for the oligonucleotidesinto the partitions, as they are capable of carrying large numbers ofoligonucleotide molecules, and may be configured to release thoseoligonucleotides upon exposure to a particular stimulus, as describedelsewhere herein. In some cases, the population of beads will provide adiverse barcode sequence library that includes at least 100 differentbarcode sequences, at least 500 different barcode sequences, at least1,000 different barcode sequences, at least 5,000 different barcodesequences, at least 10,000 different barcode sequences, at least atleast 50,000 different barcode sequences, at least 100,000 differentbarcode sequences, at least 1,000,000 different barcode sequences, atleast 5,000,000 different barcode sequences, or at least 10,000,000different barcode sequences. Additionally, each bead can be providedwith large numbers of oligonucleotide molecules attached. In particular,the number of molecules of oligonucleotides including the barcodesequence on an individual bead can be at least 100 oligonucleotidemolecules, at least 500 oligonucleotide molecules, at least 1,000oligonucleotide molecules, at least 5,000 oligonucleotide molecules, atleast 10,000 oligonucleotide molecules, at least 50,000 oligonucleotidemolecules, at least 100,000 oligonucleotide molecules, at least 500,000oligonucleotides, at least 1,000,000 oligonucleotide molecules, at least5,000,000 oligonucleotide molecules, at least 10,000,000 oligonucleotidemolecules, at least 50,000,000 oligonucleotide molecules, at least100,000,000 oligonucleotide molecules, and in some cases at least 1billion oligonucleotide molecules.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least 100 different barcode sequences, at least 500different barcode sequences, at least 1,000 different barcode sequences,at least 5,000 different barcode sequences, at least 10,000 differentbarcode sequences, at least at least 50,000 different barcode sequences,at least 100,000 different barcode sequences, at least 1,000,000different barcode sequences, at least 5,000,000 different barcodesequences, or at least 10,000,000 different barcode sequences.Additionally, each partition of the population can include at least 100oligonucleotide molecules, at least 500 oligonucleotide molecules, atleast 1,000 oligonucleotide molecules, at least 5,000 oligonucleotidemolecules, at least 10,000 oligonucleotide molecules, at least 50,000oligonucleotide molecules, at least 100,000 oligonucleotide molecules,at least 500,000 oligonucleotides, at least 1,000,000 oligonucleotidemolecules, at least 5,000,000 oligonucleotide molecules, at least10,000,000 oligonucleotide molecules, at least 50,000,000oligonucleotide molecules, at least 100,000,000 oligonucleotidemolecules, and in some cases at least 1 billion oligonucleotidemolecules.

In some cases, it may be helpful to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known barcode sequences set may provide greater assurance ofidentification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The oligonucleotides are releasable from the beads upon the applicationof a particular stimulus to the beads. In some cases, the stimulus maybe a photo-stimulus, e.g., through cleavage of a photo-labile linkagethat releases the oligonucleotides. In other cases, a thermal stimulusmay be used, where elevation of the temperature of the beads environmentwill result in cleavage of a linkage or other release of theoligonucleotides form the beads. In still other cases, a chemicalstimulus is used that cleaves a linkage of the oligonucleotides to thebeads, or otherwise results in release of the oligonucleotides from thebeads. In one case, such compositions include the polyacrylamidematrices described above for encapsulation of a cell, virus, componentsthereof, or macromolecular constituents thereof, and may be degraded forrelease of the attached oligonucleotides through exposure to a reducingagent, such as DTT.

As described herein, the cell or virus of a cell bead may include anynucleic acids within including, for example, the cell or virus's DNA,e.g., genomic DNA, RNA, e.g., messenger RNA, and the like. For example,in some cases, the methods and systems described herein are used incharacterizing expressed mRNA, including, e.g., the presence andquantification of such mRNA, and may include RNA sequencing processes asthe characterization process. Alternatively or additionally, thereagents partitioned along with the cell bead may include reagents forthe conversion of mRNA into cDNA, e.g., reverse transcriptase enzymesand reagents, to facilitate sequencing processes where DNA sequencing isemployed. Reagents may be comprised in the cell bead. Reagents may beused (e.g., used for the conversion of mRNA into cDNA) prior topartitioning. Alternatively or additionally, reagents may be usedfollowing partitioning. In some cases, where the nucleic acids to becharacterized comprise DNA, e.g., gDNA, a schematic illustration of anexample of this is shown in FIG. 3.

As shown, oligonucleotides that include a barcode sequence areco-partitioned in, e.g., a droplet 302 in an emulsion, along with asample nucleic acid 304. A sample nucleic acid may be from a cell bead.As noted elsewhere herein, the oligonucleotides 308 may be provided on abead 306 that is co-partitioned with the sample nucleic acid 304, whicholigonucleotides are releasable from the bead 306, as shown in panel A.The oligonucleotides 308 include a barcode sequence 312, in addition toone or more functional sequences, e.g., sequences 310, 314 and 316. Forexample, oligonucleotide 308 is shown as comprising barcode sequence312, as well as sequence 310 that may function as an attachment orimmobilization sequence for a given sequencing system, e.g., a P5sequence used for attachment in flow cells of an Illumina Hiseq® orMiseq® system. As shown, the oligonucleotides also include a primersequence 316, which may include a random or targeted N-mer for primingreplication of portions of the sample nucleic acid 304. Also includedwithin oligonucleotide 308 is a sequence 314 which may provide asequencing priming region, such as a “read1” or R1 priming region, thatis used to prime polymerase mediated, template directed sequencing bysynthesis reactions in sequencing systems. The functional sequences maybe selected to be compatible with a variety of different sequencingsystems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina X10,etc., and the requirements thereof. In many cases, the barcode sequence312, immobilization sequence 310 and R1 sequence 314 may be common toall of the oligonucleotides attached to a given bead. The primersequence 316 may vary for random N-mer primers, or may be common to theoligonucleotides on a given bead for certain targeted applications.

In some cases, the functional sequences may include primer sequencesuseful for RNA-seq applications. For example, in some cases, theoligonucleotides may include poly-T primers for priming reversetranscription of RNA for RNA-seq. In still other cases, oligonucleotidesin a given partition, e.g., included on an individual bead, may includemultiple types of primer sequences in addition to the common barcodesequences, such as DNA-sequencing or RNA sequencing primers, e.g.,poly-T primer sequences included within the oligonucleotides coupled tothe bead. In such cases, materials derived from a single partitionedcell bead may be subjected to DNA or RNA sequencing processes.

Based upon the presence of primer sequence 316, the oligonucleotides canprime the sample nucleic acid as shown in panel B, which allows forextension of the oligonucleotides 308 and 308 a using polymerase enzymesand other extension reagents also co-partitioned with the bead 306 andsample nucleic acid 304. As shown in panel C, following extension of theoligonucleotides that, for random N-mer primers, may anneal to multipledifferent regions of the sample nucleic acid 304; multiple overlappingcomplements or fragments of the nucleic acid are created, e.g.,fragments 318 and 320. Although including sequence portions that arecomplementary to portions of sample nucleic acid, e.g., sequences 322and 324, these constructs are generally referred to herein as comprisingfragments of the sample nucleic acid 304, having the attached barcodesequences.

The barcoded nucleic acid fragments may then be subjected tocharacterization, e.g., through sequence analysis, or they may befurther amplified in the process, as shown in panel D. For example,additional oligonucleotides, e.g., oligonucleotide 308 b, also releasedfrom bead 306, may prime the fragments 318 and 320. This is shown forfragment 318. In particular, again, based upon the presence of therandom N-mer primer 316 b in oligonucleotide 308 b (which in many casescan be different from other random N-mers in a given partition, e.g.,primer sequence 316), the oligonucleotide anneals with the fragment 318,and is extended to create a complement 326 to at least a portion offragment 318 which includes sequence 328, that comprises a duplicate ofa portion of the sample nucleic acid sequence. Extension of theoligonucleotide 308 b continues until it has replicated through theoligonucleotide portion 308 of fragment 318. As noted elsewhere herein,and as illustrated in panel D, the oligonucleotides may be configured toprompt a stop in the replication by the polymerase at a particularpoint, e.g., after replicating through sequences 316 and 314 ofoligonucleotide 308 that is included within fragment 318. As describedherein, this may be accomplished by different methods, including, forexample, the incorporation of different nucleotides and/or nucleotideanalogues that are not capable of being processed by the polymeraseenzyme used. For example, this may include the inclusion of uracilcontaining nucleotides within the sequence region 312 to prevent anon-uracil tolerant polymerase to cease replication of that region. As aresult a fragment 326 is created that includes the full-lengtholigonucleotide 308 b at one end, including the barcode sequence 312,the attachment sequence 310, the R1 primer region 314, and the randomN-mer sequence 316 b. At the other end of the sequence may be includedthe complement 316′ to the random N-mer of the first oligonucleotide308, as well as a complement to all or a portion of the R1 sequence,shown as sequence 314′. The R1 sequence 314 and its complement 314′ arethen able to hybridize together to form a partial hairpin structure 328.Because the random N-mers differ among different oligonucleotides, thesesequences and their complements may not be expected to participate inhairpin formation, e.g., sequence 316′, which is the complement torandom N-mer 316, may not be expected to be complementary to randomN-mer sequence 316 b. This may not be the case for other applications,e.g., targeted primers, where the N-mers may be common amongoligonucleotides within a given partition.

By forming these partial hairpin structures, it allows for the removalof first level duplicates of the sample sequence from furtherreplication, e.g., preventing iterative copying of copies. The partialhairpin structure also provides a useful structure for subsequentprocessing of the created fragments, e.g., fragment 326.

In general, the amplification of the nucleic acids of the cell bead maybe performed until the barcoded overlapping fragments within thepartition constitute at least 1× coverage of the particular portion orall of the associated cell or virus' genome, at least 2×, at least 3×,at least 4×, at least 5×, at least 10×, at least 20×, at least 40× ormore coverage of the genome or its relevant portion of interest. Oncethe barcoded fragments are produced, they may be directly sequenced onan appropriate sequencing system, e.g., an Illumina Hiseq®, Miseq® orX10 system, or they may be subjected to additional processing, such asfurther amplification, attachment of other functional sequences, e.g.,second sequencing primers, for reverse reads, sample index sequences,and the like.

All of the fragments from multiple different partitions may then bepooled for sequencing on high throughput sequencers as described herein,where the pooled fragments comprise a large number of fragments derivedfrom the nucleic acids of different cell beads or small cell beadpopulations, but where the fragments from the nucleic acids of a givencell bead will share the same barcode sequence. In particular, becauseeach fragment is coded as to its partition of origin, and consequentlyits single cell bead or small population of cell beads, the sequence ofthat fragment may be attributed back to that cell bead or those cellbeads (and, thus, the original cell or population of cells or viruses)based upon the presence of the barcode, which will also aid in applyingthe various sequence fragments from multiple partitions to assembly ofindividual genomes for different cell beads. This is schematicallyillustrated in FIG. 4. As shown in an example, a first nucleic acid 404from a first cell bead 400, and a second nucleic acid 406 from a secondcell bead 402 are each partitioned along with their own sets of barcodeoligonucleotides as described above. The nucleic acids may comprise achromosome, entire genome, transcript or other nucleic acid from thecell bead.

Within each partition, each cell bead's nucleic acids 404 and 406 isthen processed to separately provide overlapping set of second fragmentsof the first fragment(s), e.g., second fragment sets 408 and 410. Thisprocessing also provides the second fragments with a barcode sequencethat is the same for each of the second fragments derived from aparticular first fragment. As shown, the barcode sequence for secondfragment set 408 is denoted by “1” while the barcode sequence forfragment set 410 is denoted by “2”. A diverse library of barcodes may beused to differentially barcode large numbers of different fragment sets.However, it is not necessary for every second fragment set from adifferent first fragment to be barcoded with different barcodesequences. In fact, in many cases, multiple different first fragmentsmay be processed concurrently to include the same barcode sequence.Diverse barcode libraries are described in detail elsewhere herein.

The barcoded fragments, e.g., from fragment sets 408 and 410, may thenbe pooled for sequencing using, for example, sequence by synthesistechnologies available from Illumina or Ion Torrent division ofThermo-Fisher, Inc. Once sequenced, the sequence reads 412 can beattributed to their respective fragment set, e.g., as shown inaggregated reads 414 and 416, at least in part based upon the includedbarcodes, and in some cases, in part based upon the sequence of thefragment itself. The attributed sequence reads for each fragment set arethen assembled to provide the assembled sequence for each cell bead'snucleic acids, e.g., sequences 418 and 420, which in turn, may beattributed to individual cell beads and cell or virus (e.g., cells)encapsulated within the cell beads.

While described in terms of analyzing the genetic material presentwithin or from a cell or virus, the methods and systems described hereinmay have much broader applicability, including the ability tocharacterize other aspects of individual cells or viruses or cell orvirus populations, by allowing for the allocation of reagents toindividual cells or viruses, and providing for the attributable analysisor characterization of those cells or viruses in response to thosereagents. These methods and systems are particularly valuable in beingable to characterize a cell, virus, components thereof, ormacromolecular constituents thereof for, e.g., research, diagnostic,pathogen identification, and many other purposes.

A particularly valuable application of the cell bead processes describedherein is in the sequencing and characterization of a diseased cell thatis associated with the cell bead. A diseased cell can have alteredmetabolic properties, gene expression, and/or morphologic features.Exemplary diseases include inflammatory disorders, metabolic disorders,nervous system disorders, and cancer.

Of particular interest are cancer cells. In particular, conventionalanalytical techniques, including the ensemble sequencing processesalluded to above, are not highly adept at picking small variations ingenomic make-up of cancer cells, particularly where those exist in a seaof normal tissue cells. Further, even as between tumor cells, widevariations can exist and can be masked by the ensemble approaches tosequencing (See, e.g., Patel, et al., Single-cell RNA-seq highlightsintratumoral heterogeneity in primary glioblastoma, Science DOI:10.1126/science.1254257 (Published online Jun. 12, 2014), which isentirely incorporated herein by reference for all purposes). Cancercells may be derived from solid tumors, hematological malignancies, celllines, or obtained as circulating tumor cells, and subjected to thepartitioning processes described above. Upon analysis, one can identifyindividual cell sequences as deriving from a single cell or small groupof cells, and distinguish those over normal tissue cell sequences.

Non-limiting examples of cancer cells include cells of cancers such asAcanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginousmelanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblasticleukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia,Acute myeloblastic leukemia with maturation, Acute myeloid dendriticcell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia,Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma,Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cellleukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers,AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma,Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer,Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma,Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basalcell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma,Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma,Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer,Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Browntumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, CarcinoidTumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinomaof Unknown Primary Site, Carcinosarcoma, Castleman's Disease, CentralNervous System Embryonal Tumor, Cerebellar Astrocytoma, CerebralAstrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma,Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma,Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronicmyelogenous leukemia, Chronic Myeloproliferative Disorder, Chronicneutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectalcancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease,Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small roundcell tumor, Diffuse large B cell lymphoma, Dysembryoplasticneuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor,Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor,Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma,Epithelioid sarcoma, Erythroleukemia, Esophageal cancer,Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma,Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ CellTumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease,Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicularlymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladdercancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma,Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor,Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germcell tumor, Germinoma, Gestational choriocarcinoma, GestationalTrophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme,Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma,Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head andNeck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma,Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy,Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditarybreast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma,Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer,Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenilemyelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, KidneyCancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngealcancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and OralCavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma,Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma,Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibroushistiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma,Malignant Mesothelioma, Malignant peripheral nerve sheath tumor,Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantlecell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor,Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma,Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma,Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic SquamousNeck Cancer with Occult Primary, Metastatic urothelial carcinoma, MixedMullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor,Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiplemyeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease,Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma,Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, NasopharyngealCancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma,Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-HodgkinLymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small CellLung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma,Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer,Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer,Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor,Ovarian Low Malignant Potential Tumor, Paget's disease of the breast,Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroidcancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer,Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor ofIntermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitaryadenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonaryblastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primarycentral nervous system lymphoma, Primary effusion lymphoma, PrimaryHepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer,Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxomaperitonei, Rectal Cancer, Renal cell carcinoma, Respiratory TractCarcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma,Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygealteratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceousgland carcinoma, Secondary neoplasm, Seminoma, Serous tumor,Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome,Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor,Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Smallintestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart,Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma,Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma,Supratentorial Primitive Neuroectodermal Tumor, Surfaceepithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblasticleukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia,T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminallymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, ThymicCarcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of RenalPelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethralcancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, VaginalCancer, Verner Morrison syndrome, Verrucous carcinoma, Visual PathwayGlioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor,Wilms' tumor, and combinations thereof.

As with cancer cell analysis, the analysis and diagnosis of fetal healthor abnormality through the analysis of fetal cells is a difficult taskusing conventional techniques. In particular, in the absence ofrelatively invasive procedures, such as amniocentesis obtaining fetalcell samples can employ harvesting those cells from the maternalcirculation. Such circulating fetal cells make up an extremely smallfraction of the overall cellular population of that circulation. As aresult complex analyses are performed in order to characterize what ofthe obtained data is likely derived from fetal cells as opposed tomaternal cells. By employing the single cell characterization methodsand systems described herein, however, one can attribute genetic make upto individual cells, and categorize those cells as maternal or fetalbased upon their respective genetic make-up. Further, the geneticsequence of fetal cells may be used to identify any of a number ofgenetic disorders, including, e.g., aneuploidy such as Down syndrome,Edwards syndrome, and Patau syndrome.

Also of interest are immune cells. Methods and compositions disclosedherein can be utilized for sequence analysis of the immune repertoire.Analysis of sequence information underlying the immune repertoire canprovide a significant improvement in understanding the status andfunction of the immune system.

Non-limiting examples of immune cells which can be analyzed utilizingthe methods described herein include B cells, T cells (e.g., cytotoxic Tcells, natural killer T cells, regulatory T cells, and T helper cells),natural killer cells, cytokine induced killer (CIK) cells; myeloidcells, such as granulocytes (basophil granulocytes, eosinophilgranulocytes, neutrophil granulocytes/hypersegmented neutrophils),monocytes/macrophages, mast cell, thrombocytes/megakaryocytes, anddendritic cells. In some cases, immune cells can be analyzedindividually (i.e., as a single cell). In some cases, a single immunecell can be analyzed together with any associated pathogen (e.g.,microbe) which may be adhered to the immune cell (e.g., via an immunereceptor). In some embodiments, individual T cells are analyzed usingthe methods disclosed herein. In some embodiments, individual B cellsare analyzed using the methods disclosed herein.

Immune cells express various adaptive immunological receptors relatingto immune function, such as T cell receptors and B cell receptors. Tcell receptors and B cells receptors play a part in the immune responseby specifically recognizing and binding to antigens and aiding in theirdestruction.

The T cell receptor (TCR) is a molecule found on the surface of T cellsthat is generally responsible for recognizing fragments of antigen aspeptides bound to major histocompatibility complex (MHC) molecules. TheTCR is generally a heterodimer of two chains, each of which is a memberof the immunoglobulin superfamily, possessing an N-terminal variable (V)domain, and a C terminal constant domain. In humans, in 95% of T cellsthe TCR consists of an alpha (α) and beta (β) chain, whereas in 5% of Tcells the TCR consists of gamma and delta (γ/δ) chains. This ratio canchange during ontogeny and in diseased states as well as in differentspecies. When the TCR engages with antigenic peptide and MHC(peptide/MHC), the T lymphocyte is activated through signaltransduction.

Each of the two chains of a TCR contains multiple copies of genesegments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, anda joining “J” gene segment. The TCR alpha chain is generated byrecombination of V and J segments, while the beta chain is generated byrecombination of V, D, and J segments. Similarly, generation of the TCRgamma chain involves recombination of V and J gene segments, whilegeneration of the TCR delta chain occurs by recombination of V, D, and Jgene segments. The intersection of these specific regions (V and J forthe alpha or gamma chain, or V, D and J for the beta or delta chain)corresponds to the CDR3 region that is important for antigen-MHCrecognition. Complementarity determining regions (e.g., CDR1, CDR2, andCDR3), or hypervariable regions, are sequences in the variable domainsof antigen receptors (e.g., T cell receptor and immunoglobulin) that cancomplement an antigen. Most of the diversity of CDRs is found in CDR3,with the diversity being generated by somatic recombination eventsduring the development of T lymphocytes. A unique nucleotide sequencethat arises during the gene arrangement process can be referred to as aclonotype.

The B cell receptor, or BCR, is a molecule found on the surface of Bcells. The antigen binding portion of a BCR is composed of amembrane-bound antibody that, like most antibodies (e.g.,immunoglobulins), has a unique and randomly determined antigen-bindingsite. The antigen binding portion of a BCR includes membrane-boundimmunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, orIgE). When a B cell is activated by its first encounter with a cognateantigen, the cell proliferates and differentiates to generate apopulation of antibody-secreting plasma B cells and memory B cells. Thevarious immunoglobulin isotypes differ in their biological features,structure, target specificity and distribution. A variety of molecularmechanisms exist to generate initial diversity, including geneticrecombination at multiple sites.

The BCR is composed of two genes IgH and IgK (or IgL) coding forantibody heavy and light chains. Immunoglobulins are formed byrecombination among gene segments, sequence diversification at thejunctions of these segments, and point mutations throughout the gene.Each heavy chain gene contains multiple copies of three different genesegments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, anda joining “J” gene segment. Each light chain gene contains multiplecopies of two different gene segments for the variable region of theprotein—a variable ‘V’ gene segment and a joining ‘J’ gene segment. Therecombination can generate a molecule with one of each of the V, D, andJ segments. Furthermore, several bases may be deleted and others added(called N and P nucleotides) at each of the two junctions, therebygenerating further diversity. After B cell activation, a process ofaffinity maturation through somatic hypermutation occurs. In thisprocess progeny cells of the activated B cells accumulate distinctsomatic mutations throughout the gene with higher mutation concentrationin the CDR regions leading to the generation of antibodies with higheraffinity to the antigens. In addition to somatic hypermutation activatedB cells undergo the process of isotype switching. Antibodies with thesame variable segments can have different forms (isotypes) depending onthe constant segment. Whereas all naïve B cells express IgM (or IgD),activated B cells mostly express IgG but also IgM, IgA and IgE. Thisexpression switching from IgM (and/or IgD) to IgG, IgA, or IgE occursthrough a recombination event causing one cell to specialize inproducing a specific isotype. A unique nucleotide sequence that arisesduring the gene arrangement process can similarly be referred to as aclonotype.

In some embodiments, the methods, compositions and systems disclosedherein are utilized to analyze the various sequences of TCRs and BCRsfrom immune cells, for example various clonotypes. In some embodiments,methods, compositions and systems disclosed herein are used to analyzethe sequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain,a TCR gamma chain, or any fragment thereof (e.g., variable regionsincluding VDJ or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof). In some embodiments, methods, compositions and systemsdisclosed herein are used to analyze the sequence of a B cell receptorheavy chain, B cell receptor light chain, or any fragment thereof (e.g.,variable regions including VDJ or VJ regions, constant regions,transmembrane regions, fragments thereof, combinations thereof, andcombinations of fragments thereof).

Where immune cells are to be analyzed, primer sequences useful in any ofthe various operations for attaching barcode sequences and/oramplification reactions may comprise gene specific sequences whichtarget genes or regions of genes of immune cell proteins, for exampleimmune receptors. Such gene sequences include, but are not limited to,sequences of various T cell receptor alpha variable genes (TRAV genes),T cell receptor alpha joining genes (TRAJ genes), T cell receptor alphaconstant genes (TRAC genes), T cell receptor beta variable genes (TRBVgenes), T cell receptor beta diversity genes (TRBD genes), T cellreceptor beta joining genes (TRBJ genes), T cell receptor beta constantgenes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), Tcell receptor gamma joining genes (TRGJ genes), T cell receptor gammaconstant genes (TRGC genes), T cell receptor delta variable genes (TRDVgenes), T cell receptor delta diversity genes (TRDD genes), T cellreceptor delta joining genes (TRDJ genes), and T cell receptor deltaconstant genes (TRDC genes).

The ability to characterize individual cells, viruses, componentsthereof, or macromolecular constituents thereof from larger diversepopulations of these entities is also of significant value in bothenvironmental testing as well as in forensic analysis, where samplesmay, by their nature, be made up of diverse populations of cells orviruses and other material that “contaminate” the sample, relative tothe cell(s) or virus(es) for which the sample is being tested, e.g.,environmental indicator organisms, toxic organisms, and the like for,e.g., environmental and food safety testing, victim and/or perpetratorcells in forensic analysis for sexual assault, and other violent crimes,and the like.

Additional useful applications of the above described cell beadsequencing and characterization processes are in the field ofneuroscience research and diagnosis. In particular, neural cells caninclude long interspersed nuclear elements (LINEs), or ‘jumping’ genesthat can move around the genome, which cause each neuron to differ fromits neighbor cells. Research has shown that the number of LINEs in humanbrain exceeds that of other tissues, e.g., heart and liver tissue, withbetween 80 and 300 unique insertions (See, e.g., Coufal, N. G. et al.Nature 460, 1127-1131 (2009), which is entirely incorporated herein byreference for all purposes). These differences have been postulated asbeing related to a person's susceptibility to neuro-logical disorders(see, e.g., Muotri, A. R. et al. Nature 468, 443-446 (2010), which isentirely incorporated herein by reference for all purposes), or providethe brain with a diversity with which to respond to challenges. As such,the methods described herein may be used in the sequencing andcharacterization of individual neural cells.

The cell bead analysis methods described herein are also useful in theanalysis of gene expression, as noted above, both in terms ofidentification of RNA transcripts and their quantitation. In particular,using the single cell level analysis methods described herein, one canisolate and analyze the RNA transcripts present in individual cells orviruses, populations of cells or viruses, or subsets of populations ofcells or viruses. In particular, in some cases, the barcodeoligonucleotides may be configured to prime, replicate and consequentlyyield barcoded fragments of RNA from individual cells or viruses. Forexample, in some cases, the barcode oligonucleotides may include mRNAspecific priming sequences, e.g., poly-T primer segments that allowpriming and replication of mRNA in a reverse transcription reaction orother targeted priming sequences. Alternatively or additionally, randomRNA priming may be performed using random N-mer primer segments of thebarcode oligonucleotides. Methods for RNA, mRNA and cell featureanalysis are provided in U.S. Patent Publication No. 2015/0376609, whichis entirely incorporated herein by reference.

In some cases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method. In a PHASE method, a randomN-mer sequence may be used to randomly prime a sample, such as genomicDNA (gDNA). In some embodiments, the random N-mer may comprise a primer.In some cases, the random N-mer may prime a sample. In some cases, therandom N-mer may prime genomic DNA. In some cases, the random N-mer mayprime DNA fragments. An example PHASE method is shown schematically inFIG. 3. Additional examples of PHASE are provided in U.S. PatentPublication No. 2014/0378345, which is entirely incorporated herein byreference.

Additionally, a random N-mer sequence may also be attached to anotheroligonucleotide. This oligonucleotide may be a universal sequence and/ormay contain one or more primer read sequences that may be compatiblewith a sequencing device (e.g. Read 1 primer site, Read 2 primer site,Index primer site), one or more barcode sequences, and one or moreadaptor segments that may be compatible with a sequencing device (e.g.P5, P7). Alternatively, the oligonucleotide may comprise none of theseand may include another sequence.

Via subsequent amplification methods, priming of a sample nucleic acidwith a random N-mer may be used to attach an oligonucleotide sequence(e.g., an oligonucleotide sequence comprising a barcode sequence) linkedto a random N-mer to the sample nucleic acid, including a sample nucleicacid to be sequenced. Utilizing random primers to prime a sample mayintroduce significant sequence read errors, due to, for example, theproduction of undesired amplification products. An example PHASE methodis shown schematically in FIG. 3. Additional examples of PHASE areprovided in U.S. Patent Publication No. 2014/0378345, which is entirelyincorporated herein by reference.

To mitigate undesired amplification products, at least a subsection ofan oligonucleotide sequence (e.g., an oligonucleotide comprising aprimer) used for PHASE amplification may be substituted withuracil-containing nucleotides in place of thymine containingnucleotides, respectively. In some cases, substitution may be complete(e.g., all thymine containing nucleotides are substituted with uracilcontaining nucleotides), or may be partial such that a portion of anoligonucleotide's thymine containing nucleotides are substituted withuracil containing nucleotides. In some cases, thymine containingnucleotides in all but the last about 10 to 20, last about 10 to 30,last about 10 to 40, or last about 5 to 40 nucleotides of anoligonucleotide sequence adjacent to a random N-mer sequence aresubstituted with uracil containing nucleotides, or functionalequivalents thereof. In addition, a polymerase that does not accept orprocess uracil-containing templates may be used for amplification of thesample nucleic acid. In this case, the non-uracil containing portion ofabout 10 to about 20 nucleotides may be amplified and the remainingportion containing uracil containing nucleotides may not be amplified.In some cases, the portion of an oligonucleotide sequence comprisinguracil containing nucleotides may be adjacent to the N-mer sequence. Insome cases, the portion of an oligonucleotide sequence comprising uracilcontaining nucleotides may be adjacent to the barcode sequence. Anyportion of an oligonucleotide sequence, including an adaptor segment,barcode, or read primer sequence may comprise uracil containingnucleotides (e.g., substituted for thymine containing nucleotides),depending upon the configuration of the oligonucleotide sequence. Insome cases, uracil containing nucleotides can be introduced tooligonucleotides during PHASE amplification with the inclusion of dUTPnucleotides in place of or in combination with dTTPs in amplificationreactions.

The dUTP concentration may be increased over time. For instance, thedUTP concentration may be increased at a controlled rate by theinclusion of dCTP deaminase in an amplification reaction mixture. ThedUTP concentration may be increased over time by the dCTP-mediatedconversion of dCTP into dUTP. This may result in an increasedincorporation of dUTP into daughter DNA fragments. The uracil bases maybe excised. As the dUTP concentration increases over the course of areaction, the reaction products may become shorter and thus availablefor barcoding. The dCTP aminase activity may be modified by adjustingthe reaction parameters. For instance, the dCTP aminase activity may bemodified by altering the reaction temperature, pH, dCTP concentration,inorganic phosphate concentration, and/or dTTP concentration. The dUTPconcentration may also be modified by the production of dUTP in thereaction mixture. For instance, the reaction may be supplied withdeoxycytidine monophosphate (dCMP) or deoxycytidine diphosphate (dCDP).A deaminase and/or kinase may then act upon the dCMP or dCDP to producedUTP.

In some cases, a plurality of targeted constructs comprising a barcodesequence and a targeted N-mer comprising a poly-T sequence may becoupled to a bead (e.g., a gel bead). In some cases, the plurality ofconstructs may comprise an identical barcode sequence. The beads may bepartitioned (e.g., in fluidic droplets) with sample nucleic acidcomprising RNA, the bead(s) in each partition degraded to release thecoupled constructs into the partition, and the sample RNA captured viathe targeted N-mer of the constructs. Partitions may also comprisebarcode constructs (e.g., with barcode sequences identical to thetargeted constructs) that comprise a random N-mer. In a firstamplification cycle, extension of the targeted constructs can occur viareverse transcription within each partition, to generate extensionproducts comprising the targeted construct. The extension products ineach partition can then be primed with the barcode constructs comprisingthe random N-mer to generate partial hairpin amplicons as describedabove. Post processing (e.g., addition of additional sequences (e.g.,P7, R2), addition of a sample index, etc.) of the generated ampliconsmay be achieved with any method described herein, including bulkamplification methods (e.g., bulk PCR) and bulk ligation.

In some cases, reverse transcription of RNA in a sample may also be usedwithout the use of a targeted barcode construct. For example, samplenucleic acid comprising RNA may be first subject to a reversetranscription reaction with other types of reverse transcription primerssuch that cDNA is generated from the RNA. The cDNA that is generated maythen undergo targeted or non-targeted amplification as described herein.For example, sample nucleic acid comprising RNA may be subject to areverse transcription reaction such that cDNA is generated from the RNA.The cDNA may then enter a PHASE amplification reaction, using a barcodeconstruct with a random N-mer as described above, to generate partialhairpin amplicons comprising the construct's barcode sequence. Postprocessing (e.g., addition of additional sequences (e.g., P7, R2),addition of a sample index, etc.) of the generated partial hairpinamplicons may be achieved with any method described herein, includingbulk amplification methods (e.g., bulk PCR) and bulk ligation.

Targeted barcode constructs may also be generated toward specificsequences (e.g., gene sequences) on specific strands of a nucleic acidsuch that strandedness information is retained for sequencer-readyproducts generated for each strand. For example, a sample nucleic maycomprise double stranded nucleic acid (e.g., double-stranded DNA), suchthat each strand of nucleic acid comprises one or more different targetgene sequences. Complementary DNA strands can comprise different genesequences due to the opposite 5′ to 3′ directionalities and/or basecomposition of each strand. Targeted barcode constructs can be generatedfor each strand (based on 5′ to 3′ directionality of the strand) basedon the targeted N-mer and configuration of the barcode construct.

A first and second set of targeted barcode constructs may be targeted toeither of a forward strand and reverse strand of a double-strandedsample nucleic acid. The first set can comprise targeted barcodeconstructs comprising a P5 sequence, a barcode sequence, and a targetedN-mer to either of a first target sequence or a second target sequence.The second set can comprise targeted barcode constructs comprising a P5sequence, a barcode sequence, and a targeted N-mer to either of thefirst target sequence and the second target sequence. Each construct canalso comprise any additional sequences between the barcode and thetargeted N-mer.

The barcode constructs in the first set can be configured to prime theirrespective target sequences on the forward strand of the double-strandedsample nucleic acid. The barcode constructs of the second set can beconfigured to prime their respective target sequences on the reversestrand of the double-stranded sample nucleic acid. The targeted barcodeconstructs in each set can be configured in opposite directionalitycorresponding to the opposite directionality of forward and reversestrands of the double-stranded sample nucleic acid. Each barcodeconstruct can prime its respective target sequence on its respectivestrand of sample nucleic acid to generate barcoded amplicons via anamplification reaction, such as any amplification reaction describedherein.

Additional sequences can be added to barcoded amplicons usingamplification methods described herein, including bulk amplification,bulk ligation, or a combination thereof. A first primer set correspondsto the first targeted barcode construct set and a second primer setcorresponds to the second targeted barcode construct set. Each primercan prime its respective target sequence on its respective strand andbulk amplification (e.g., bulk PCR) initiated to generatesequencer-ready constructs that include the P7 and sample indexsequences in analogous fashion to bulk amplification methods describedelsewhere herein. Based on the configuration and directionality of thevarious components of each sequencer-ready construct (e.g., P5, barcode,targeted N-mer, sample insert, etc.), the strand from which thesequencer-ready product is generated can be determined/is retained.

Methods described herein may be useful in whole genome amplification. Insome embodiments of whole genome amplification, a random primer (e.g., arandom N-mer sequence) can be hybridized to a genomic nucleic acid. Therandom primer can be a component of a larger oligonucleotide that mayalso include a universal nucleic acid sequence (including any type ofuniversal nucleic acid sequence described herein) and a nucleic acidbarcode sequence. In some cases, the universal nucleic acid sequence maycomprise one or more uracil containing nucleotides. Moreover, in somecases, the universal nucleic acid sequence may comprise a segment of atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more nucleotides that do not comprise uracil. The random primercan be extended (e.g., in a primer extension reaction or any othersuitable type of nucleic acid amplification reaction) to form anamplified product.

In some embodiments of whole genome amplification, a genomic component(e.g., a chromosome, genomic nucleic acid such as genomic DNA, a wholegenome of an organism, or any other type of genomic component describedherein) may be fragmented in a plurality of first fragments. The firstfragments can be co-partitioned into a plurality of partitions with aplurality of oligonucleotides. The oligonucleotides in each of thepartitions may comprise a primer sequence (including a type of primersequence described elsewhere herein) and a common sequence (e.g., abarcode sequence). Primer sequences in each partition can then beannealed to a plurality of different regions of the first fragmentswithin each partition. The primer sequences can then be extended alongthe first fragments to produce amplified first fragments within eachpartition of the plurality of partitions. The amplified first fragmentswithin the partitions may comprise any suitable coverage (as describedelsewhere herein) of the genomic component. In some cases, the amplifiedfirst fragments within the partitions may comprise at least 1× coverage,at least 2× coverage, at least 5× coverage, at least 10× coverage, atleast 20× coverage, at least 40× coverage, or greater coverage of thegenomic component.

In some examples, amplification is performed using methods disclosed inU.S. Patent Application Publication No. 2016/0257984, which is entirelyincorporated herein by reference for all purposes. In some cases,amplification may be performed using a priming free amplification bypolymerization at nick sites (such as the priming free polymerizationmethods disclosed in U.S. Patent Application Publication No.2016/0257984, which is entirely incorporated herein by reference for allpurposes). Sequencing libraries produced via priming free amplificationmay provide superior sequencing results when compared to conventionalprimer-based amplification library preparation approaches. For instance,the priming free amplification approach may result in more evensequencing coverage across a broad range of GC base content whencompared to primer-based amplification results. Improved sequencingcoverage evenness may be achieved in priming free amplification,resulting in a more Poissonian distribution when compared to thedistributions achieved using primer-based amplification.

FIG. 21 illustrates the process of library preparation using primingfree amplification of templates. Although illustrated as a series ofpanels in FIG. 21, the reaction processes illustrated may be performedsimultaneously with all the reagents present together in the reactionmixture during the priming free amplification by polymerization process.This process may be contrasted with a standard primed amplificationprocess for preparing a sequencing library.

At (i) in FIG. 21, a DNA polymerase, such as phi29 DNA Polymerase (NewEngland Biolabs ® Inc. (NEB), Ipswich, Mass.), may be used to performisothermal amplification. The isothermal amplification may compriseinitiation using a hexamer (short arrow) and phi29 DNA polymerase (oval)which has very high processivity and fidelity that may result in evencoverage and low error rates. As the polymerase processes along thetarget sequence (long line), a copied DNA template is produced. In thepresence of all deoxyribonucleotide triphosphates (nucleotides) and asmall amount of deoxyribouracil triphosphate, the polymerase basedincorporation of dUTP results in a growing template strand (long arrow)at (ii) in FIG. 21. The reaction may include an enzyme (oval with bolt)capable of excising dUTP and creating nicks in the copied template DNAstrand, but not in the original target sequence. At (iii) in FIG. 21,the nicking by the enzyme capable of excising dUTP may result in theproduction of a plurality of amplified strands (short arrows), each ofwhich may be shorter than the original template strand. Additionally,phi29 DNA polymerase may engage at the nick sites for additionalamplification in a priming independent amplification process. At (v) inFIG. 21, the original target sequence may be recycled as a template uponstrand displacement of released amplified fragments owing to the highlyprocessive nature of the phi29 DNA polymerase. Subsequent amplificationsmay mirror the previously described process to produce additionalreleased amplified fragments.

The priming free amplification methods may be extended to provide abarcoding capability, for instance as shown in FIGS. 22A-C.

FIG. 22A shows a method of barcoding amplified templates generated bythe priming free amplification using an extension barcoding approach.Strand displacement and the high processivity of phi29 DNA polymerasemay allow the release of amplified fragments, thereby enabling recyclingof the template for further amplification. The single strand fragmentsthat are generated during stand displacement may be converted to dsDNAby the hexamer or by the Nmer part of the same polymerase.

FIG. 22B shows a method of barcoding amplified templates generated bythe priming free amplification using a single stranded or doublestranded template to barcode ligation approach. The template DNAmolecules may be converted to either single stranded (using, forinstance, changes in temperature or an enzyme) or double stranded(using, for instance, an enzyme). The molecular barcodes, (such asoligonucleotides) may be attached through a ligation process using assDNA ligase, dsDNA ligase, or another nucleic acid modifying enzyme.Additional oligonucleotides serving as molecular handles may be added tothe first barcode tag in subsequent ligations.

FIG. 22C shows a method of barcoding amplified templates generated bythe priming free amplification by attaching a single strand DNA molecule(with barcode or primer sequence) to a bead from the 3′ end. The 5′ endof the oligo may be pre-adenylated (either chemically or enzymatically).The oligo may be sequestered using Hotstart-IT binding protein which maybe released using heat. For barcoding the single-stranded librarymolecules (single strands generated by heat treatment or helicase), APPDNA/RNA ligase may ligate 5′ pre-adenylated oligo with 3′ end of thelibrary molecule. This process may be very specific, as oligo-oligoligation may be avoided by blocking the 3′ end. Library molecules may beunable to self-ligate as they are not adenlyated. The APP DNA/RNA ligasemay be a thermostable 5′ App DNA/RNA Ligase including a point mutant ofcatalytic lysine of RNA ligase from Methanobacteriumthermoautotrophicum. This enzyme may be ATP independent. It may requirea 5′ pre-adenylated linker for ligation to the 3′-OH end of either RNAor single stranded DNA (ssDNA).

A further approach to molecular barcoding following the priming freeamplification is the use of a topoisomerase enzyme. For instance,topoisomerase I from Vaccinia virus may bind to duplex DNA at specificsites and cleave the phosphodiester backbone after 5′-CCCTT in onestrand. Molecular barcoding may be achieved when an adapter sequence(such as an oligonucleotide) is pre-bound to a topoisomerase enzyme. Theamplified templates may be prepared for blunt end ligation using, forinstance, the Klenow fragment of DNA polymerase.

In some cases, amplification may be performed using the degenerateoligonucleotide primed-polymerase chain reaction (DOP-PCR) method.DOP-PCR uses a partially degenerate sequence in a PCR protocol with twodifferent annealing temperatures. The first PCR cycles are performedusing a low annealing temperature. These cycles are then followed by alarge number of PCR cycles with a higher annealing temperature. The useof the lower first annealing temperature may ensure that fragments thatare specifically tagged in the first PCR cycles are amplified at thehigher second annealing temperature. The DOP-PCR method may allow randomamplification of DNA from any source.

In addition to the use of two annealing temperatures, DOP-PCR ischaracterized by the use of modified PCR primers. The DOP-PCR primerconsists of three regions. The 5′-end carries a recognition sequence forXhoI (C⋅TCGAG), a restriction endonuclease that cuts rarely within thehuman genome. The sequence is then followed by a middle portioncontaining six nucleotides of degenerate sequence (NNNNNN, where N=A, C,G, or T in approximately equal proportions) and a 3′-end sequencecontaining six specific bases (ATGTGG) which primes the reactionapproximately every 4 kb. At a sufficiently low annealing temperaturethe six specific nucleotides included in the 3′-end of the degenerateoligonucleotide will anneal to the genomic strand allowing the primer toinitiate PCR. The PCR fragments are then generated which contain thefull length of the oligoprimer at one end and its complementary sequenceat the other end. Subsequently, the temperature is increased to thelevel required for the full length of the degenerate primer to anneal.

In contrast to the pairs of target-specific primer sequences used intraditional PCR, a single primer, which has defined sequences at its5′-end (containing an XhoI restriction site) and 3′-end and a randomhexamer sequence between them, is used here. DOP-PCR comprises twodifferent cycling stages. In the first low stringency phase,low-temperature annealing and extension in the first five to eightcycles occurs at many binding sites in the genome. The 3′-end of theprimer binds at sites in the genome complementary to the 6-bpwell-defined sequence at the 3′-end of the primer (˜10(6) sites in thehuman genome). The adjacent random hexamer sequence (displaying allpossible combinations of the nucleotides A, G, C, and T) can then annealand tags these sequences with the DOP primer. In the second stage, thePCR annealing temperature is raised, which increases priming specificityduring amplification of the tagged sequence.

Additional examples of the DOP-PCR method are provided, for example, inArneson et al, Whole-genome amplification by degenerate oligonucleotideprimed PCR (DOP-PCR), CSH Protoc DOI: 10.1101/pdb.prot4919 (PublishedJan. 1, 2008), which is entirely incorporated herein by reference forall purposes.

Although operations with various barcode designs have been discussedindividually, individual beads can include barcode oligonucleotides ofvarious designs for simultaneous use.

In addition to characterizing individual cells or viruses or cell orvirus sub-populations from larger populations, the processes and systemsdescribed herein may also be used to characterize individual cells orviruses as a way to provide an overall profile of a cellular, or otherorganismal population. A variety of applications require the evaluationof the presence and quantification of different cells or viruses ororganism types within a population of cells or viruses, including, forexample, microbiome analysis and characterization, environmentaltesting, food safety testing, epidemiological analysis, e.g., in tracingcontamination or the like. In particular, the analysis processesdescribed above may be used to individually characterize, sequenceand/or identify large numbers of individual cells or viruses within apopulation. This characterization may then be used to assemble anoverall profile of the originating population, which can provideimportant prognostic and diagnostic information.

For example, shifts in human microbiomes, including, e.g., gut, buccal,epidermal microbiomes, etc., have been identified as being bothdiagnostic and prognostic of different conditions or general states ofhealth. Using the cell bead analysis methods and systems describedherein, one can again, characterize, sequence and identify individualcells in an overall population, and identify shifts within thatpopulation that may be indicative of diagnostic ally relevant factors.By way of example, sequencing of bacterial 16S ribosomal RNA genes hasbeen used as a highly accurate method for taxonomic classification ofbacteria. Using the targeted amplification and sequencing processesdescribed above can provide identification of individual cells within apopulation of cells. One may further quantify the numbers of differentcells within a population to identify current states or shifts in statesover time. See, e.g., Morgan et al, PLoS Comput. Biol., Ch. 12, December2012, 8(12):e1002808, and Ram et al., Syst. Biol. Reprod. Med., June2011, 57(3):162-170, each of which is entirely incorporated herein byreference for all purposes. Likewise, identification and diagnosis ofinfection or potential infection may also benefit from the cell beadanalyses described herein, e.g., to identify microbial species presentin large mixes of other cells and/or nucleic acids, from anydiagnostically relevant environment, e.g., cerebrospinal fluid, blood,fecal or intestinal samples, or the like.

The foregoing analyses may also be particularly useful in thecharacterization of potential drug resistance of different cells orpathogens, e.g., cancer cells, bacterial pathogens, etc., through theanalysis of distribution and profiling of different resistancemarkers/mutations across cell populations in a given sample.Additionally, characterization of shifts in these markers/mutationsacross populations of cells over time can provide valuable insight intothe progression, alteration, prevention, and treatment of a variety ofdiseases characterized by such drug resistance issues.

Similarly, analysis of different environmental samples to profile themicrobial organisms, viruses, or other biological contaminants that arepresent within such samples, can provide important information aboutdisease epidemiology, and potentially aid in forecasting diseaseoutbreaks, epidemics an pandemics.

As described above, the methods, systems and compositions describedherein may also be used for analysis and characterization of otheraspects of individual cells or viruses or populations of cells orviruses. In an example process, a sample is provided that contains cellsassociated with cell beads that are to be analyzed and characterized asto their cell surface proteins. Also provided is a library ofantibodies, antibody fragments, or other molecules having a bindingaffinity to the cell surface proteins or antigens (or other cellfeatures) for which the cell is to be characterized (also referred toherein as cell surface feature binding groups). For ease of discussion,these affinity groups are referred to herein as binding groups. Thebinding groups can include a reporter molecule that is indicative of thecell surface feature to which the binding group binds. In particular, abinding group type that is specific to one type of cell surface featurewill comprise a first reporter molecule, while a binding group type thatis specific to a different cell surface feature will have a differentreporter molecule associated with it. In some aspects, these reportermolecules will comprise oligonucleotide sequences. Oligonucleotide basedreporter molecules can provide advantages of being able to generatesignificant diversity in terms of sequence, while also being readilyattachable to most biomolecules, e.g., antibodies, etc., as well asbeing readily detected, e.g., using sequencing or array technologies. Inthe example process, the binding groups include oligonucleotidesattached to them. Thus, a first binding group type, e.g., antibodies toa first type of cell surface feature, will have associated with it areporter oligonucleotide that has a first nucleotide sequence. Differentbinding group types, e.g., antibodies having binding affinity for other,different cell surface features, will have associated therewith reporteroligonucleotides that comprise different nucleotide sequences, e.g.,having a partially or completely different nucleotide sequence. In somecases, for each type of cell surface feature binding group, e.g.,antibody or antibody fragment, the reporter oligonucleotide sequence maybe known and readily identifiable as being associated with the knowncell surface feature binding group. These oligonucleotides may bedirectly coupled to the binding group, or they may be attached to abead, molecular lattice, e.g., a linear, globular, cross-slinked, orother polymer, or other framework that is attached or otherwiseassociated with the binding group, which allows attachment of multiplereporter oligonucleotides to a single binding group.

In the case of multiple reporter molecules coupled to a single bindinggroup, such reporter molecules can comprise the same sequence, or aparticular binding group will include a known set of reporteroligonucleotide sequences. As between different binding groups, e.g.,specific for different cell surface features, the reporter molecules canbe different and attributable to the particular binding group.

Attachment of the reporter groups to the binding groups may be achievedthrough any of a variety of direct or indirect, covalent or non-covalentassociations or attachments. For example, in the case of oligonucleotidereporter groups associated with antibody based binding groups, sucholigonucleotides may be covalently attached to a portion of an antibodyor antibody fragment using chemical conjugation techniques (e.g.,Lightning-Link® antibody labeling kits available from InnovaBiosciences), as well as other non-covalent attachment mechanisms, e.g.,using biotinylated antibodies and oligonucleotides (or beads thatinclude one or more biotinylated linker, coupled to oligonucleotides)with an avidin or streptavidin linker. Antibody and oligonucleotidebiotinylation techniques are available (See, e.g., Fang, et al.,Fluoride-Cleavable Biotinylation Phosphoramidite for 5′-end-Labeling andAffinity Purification of Synthetic Oligonucleotides, Nucleic Acids Res.Jan. 15, 2003; 31(2):708-715, DNA 3′ End Biotinylation Kit, availablefrom Thermo Scientific, which is entirely incorporated herein byreference for all purposes). Likewise, protein and peptide biotinylationtechniques have been developed and are readily available (See, e.g.,U.S. Pat. No. 6,265,552, which is entirely incorporated herein byreference for all purposes).

The reporter oligonucleotides may be provided having any of a range ofdifferent lengths, depending upon the diversity of reporter molecules ora given analysis, the sequence detection scheme employed, and the like.In some cases, these reporter sequences can be greater than about 5nucleotides in length, greater than or equal to about 10 nucleotides inlength, greater than or equal to about 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 150 or 200 nucleotides in length. In some cases, thesereporter nucleotides may be less than about 250 nucleotides in length,less than or equal to about 200, 180, 150, 120 100, 90, 80, 70, 60, 50,40, or 30 nucleotides in length. In many cases, the reporteroligonucleotides may be selected to provide barcoded products that arealready sized, and otherwise configured to be analyzed on a sequencingsystem. For example, these sequences may be provided at a length thatideally creates sequenceable products of a length for particularsequencing systems. Likewise, these reporter oligonucleotides mayinclude additional sequence elements, in addition to the reportersequence, such as sequencer attachment sequences, sequencing primersequences, amplification primer sequences, or the complements to any ofthese.

In operation, a cell-containing sample is incubated with the bindingmolecules and their associated reporter oligonucleotides, for any of thecell surface features to be analyzed. Following incubation, the cellsare washed to remove unbound binding groups. Following washing, thecells (or components) are encapsulated into cell beads and the cellbeads partitioned into separate partitions, e.g., droplets, along withthe barcode carrying beads described above, where each partitionincludes a limited number of cells, e.g., in some cases, a single cell.Upon releasing the barcodes from the beads and the cell or cellcomponents from the cell beads, they will prime the amplification andbarcoding of the reporter oligonucleotides. As noted above, the barcodedreplicates of the reporter molecules may additionally include functionalsequences, such as primer sequences, attachment sequences or the like.

The barcoded reporter oligonucleotides are then subjected to sequenceanalysis to identify which reporter oligonucleotides bound to the cellswithin the partitions. Further, by also sequencing the associatedbarcode sequence, one can identify that a given cell surface featurelikely came from the same cell as other, different cell surfacefeatures, whose reporter sequences include the same barcode sequence,i.e., they were derived from the same partition.

Based upon the reporter molecules that emanate from an individualpartition based upon the presence of the barcode sequence, one may thencreate a cell surface profile of individual cells from a population ofcells. Profiles of individual cells or populations of cells may becompared to profiles from other cells, e.g., ‘normal’ cells, to identifyvariations in cell surface features, which may provide diagnosticallyrelevant information. In particular, these profiles may be particularlyuseful in the diagnosis of a variety of disorders that are characterizedby variations in cell surface receptors, such as cancer and otherdisorders.

In one application, the methods and systems described herein may be usedto characterize cell or virus features, such as cell surface features,e.g., proteins, receptors, etc. In particular, the methods describedherein may be used to attach reporter molecules to these cell features,that when partitioned as described above, may be barcoded and analyzed,e.g., using DNA sequencing technologies, to ascertain the presence, andin some cases, relative abundance or quantity of such cell or virusfeatures within an individual cell or virus or population of cells orviruses.

In a particular example, a library of potential cell binding ligands,e.g., antibodies, antibody fragments, cell surface receptor bindingmolecules, or the like, maybe provided associated with a first set ofnucleic acid reporter molecules, e.g., where a different reporteroligonucleotide sequence is associated with a specific ligand, andtherefore capable of binding to a specific cell surface feature. In someaspects, different members of the library may be characterized by thepresence of a different oligonucleotide sequence label, e.g., anantibody to a first type of cell surface protein or receptor may haveassociated with it a first known reporter oligonucleotide sequence,while an antibody to a second receptor protein may have a differentknown reporter oligonucleotide sequence associated with it. Prior toco-partitioning, the cells may be incubated with the library of ligands,that may represent antibodies to a broad panel of different cell surfacefeatures, e.g., receptors, proteins, etc., and which include theirassociated reporter oligonucleotides. Unbound ligands are washed fromthe cells, and the cells are then co-partitioned along with the barcodeoligonucleotides described above. As a result, the partitions willinclude the cell or cells, as well as the bound ligands and their known,associated reporter oligonucleotides.

One may then subject the reporter oligonucleotides to the barcodingoperations described above for cellular nucleic acids, to producebarcoded, reporter oligonucleotides, where the presence of the reporteroligonucleotides can be indicative of the presence of the particularcell surface feature, and the barcode sequence will allow theattribution of the range of different cell surface features to a givenindividual cell or population of cells based upon the barcode sequencethat was co-partitioned with that cell or population of cells. As aresult, one may generate a cell-by-cell profile of the cell surfacefeatures within a broader population of cells. This aspect of themethods and systems described herein is described in greater detailbelow.

This example is schematically illustrated in FIG. 5. As shown, apopulation of cells, represented by cells or cell components 502 and 504are incubated with a library of cell surface associated reagents, e.g.,antibodies, cell surface binding proteins, ligands or the like, whereeach different type of binding group includes an associated nucleic acidreporter molecule associated with it, shown as ligands and associatedreporter molecules 506, 508, 510 and 512 (with the reporter moleculesbeing indicated by the differently shaded circles). Where the cellexpresses the surface features that are bound by the library, theligands and their associated reporter molecules can become associated orcoupled with the cell surface. Individual cells are encapsulated intocell beads, in some cases subject to lysis and/or denaturing conditions,and the resulting cell beads are then partitioned into separatepartitions, e.g., droplets 514 and 516, along with their associatedligand/reporter molecules, as well as an individual barcodeoligonucleotide bead as described elsewhere herein, e.g., beads 522 and524, respectively. The cellular material is released from the cell beadsand the barcoded oligonucleotides are released from the beads and usedto attach the barcode sequence the reporter molecules present withineach partition with a barcode that is common to a given partition, butwhich varies widely among different partitions. For example, as shown inFIG. 5, the reporter molecules that associate with cell or cellcomponents 502 in partition 514 are barcoded with barcode sequence 518,while the reporter molecules associated with cell or cell components 504in partition 516 are barcoded with barcode 520. As a result, one isprovided with a library of oligonucleotides that reflects the surfaceligands of the cell, as reflected by the reporter molecule, but which issubstantially attributable to an individual cell by virtue of a commonbarcode sequence, allowing a single cell level profiling of the surfacecharacteristics of the cell. This process is not limited to cell surfacereceptors but may be used to identify the presence of a wide variety ofspecific cell structures, chemistries or other characteristics. Cellbead processing and analysis methods and systems described herein can beutilized for a wide variety of applications, including analysis ofspecific individual cells, analysis of different cell types withinpopulations of differing cell types, analysis and characterization oflarge populations of cells for environmental, human health,epidemiological forensic, or any of a wide variety of differentapplications.

Cells may be treated with cell surface associated reagents prior tobeing processed such that the cells or components of the cells areencapsulated within cell beads. Upon partitioning of cell beads withbarcoded beads as described elsewhere herein, barcodes from the barcodebeads can be used to generate barcoded constructs derived from reportermolecules associated with cell surface associated reagents.

Also provided herein are kits for analyzing individual cells or virusesor small populations of cells or viruses. The kits may include one, two,three, four, five or more, up to all of partitioning fluids, includingboth aqueous buffers and non-aqueous partitioning fluids or oils,nucleic acid barcode libraries that are releasably associated withbeads, as described herein, microfluidic devices, reagents fordisrupting cells amplifying nucleic acids, and providing additionalfunctional sequences on fragments of cellular nucleic acids orreplicates thereof, as well as instructions for using any of theforegoing in the methods described herein.

In encapsulating single cell beads and single barcode beads within adroplet, it may be useful to utilize methods and systems which allow oneor more chemical or biochemical operations enacted on the encapsulatedmaterial of the single cell bead to proceed to completion prior toallowing the encapsulated material to interact with the barcodes of thebarcode bead. For instance, chemicals used in preparing a cell forbarcoding may be chemically incompatible with the beads or barcodesthemselves. As an example, prior to or contemporaneous toco-partitioning cell beads and barcode beads, lysis agents (which, may,for example, degrade barcodes), such as sodium hydroxide (NaOH), may beused to lyse a cell encapsulated in a cell bead in order to allow themacromolecular constituents of the encapsulated be released for laterinteraction with the bead and its barcodes.

Furthermore, reagents may be used to perform one or more additionalchemical or biochemical operation following lysis of a cell encapsulatedin a cell bead. Reagents may include any reagents useful in performingan operation (e.g., a reaction), such as, for example, nucleic acidmodification (e.g., ligation, digestion, methylation, randommutagenesis, bisulfite conversion, uracil hydrolysis, nucleic acidrepair, capping, or decapping), nucleic acid amplification (e.g.,isothermal amplification or PCR), nucleic acid insertion or cleavage(e.g., via CRISPR/Cas9-mediated or transposon-mediated insertion orcleavage), or reverse transcription. Additionally, it may be useful toutilize methods and systems that allow the preparation of targetsequence or sequencing reads specific to macromolecular constituents ofinterest at a higher rate than non-target specific reads. For instance,the methods and systems may be characterized by their suppression of notemplate control (NTC) effects.

The systems and methods described herein may allow for the production ofone or more droplets containing a single cell bead and a single barcodebead. The systems and methods may also allow for the production of oneor more droplets containing a single cell bead and more than barcode onebead, one or more droplets containing more than one cell bead and asingle barcode bead, or one or more droplets containing more than onecell bead and more than one barcode bead.

FIG. 7 shows a flowchart for a method 700 of producing dropletscontaining a cell bead and a barcode bead (e.g., gel bead) comprising abarcode sequence and generating sequence reads from macromolecularcomponents of the cell bead.

In operation 710, a first liquid phase comprising a plurality of cellbeads is provided. The first liquid phase may be aqueous. The firstliquid phase may comprise a cellular growth medium. The first liquidphase may comprise a minimal growth medium.

In operation 720, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.The second liquid phase may comprise a cellular growth medium. Thesecond liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of cell beads. In some cases, the firstliquid phase and the second liquid phase are the same phase. In somecases, the first liquid phase and the second liquid phase are mixed toprovide a mixed phase.

In operation 730, the first liquid phase and the second liquid phase canbe brought together with a third liquid phase that is immiscible withthe first and second liquid phase. The third liquid phase may interactwith the first and second liquid phases in such a manner as to partitioneach of the plurality of cell beads and the plurality of barcode beadsinto a plurality of droplets. The third liquid phase may comprise anoil. The third liquid phase may comprise a fluorinated hydrocarbon. Insome cases, a given droplet may include a single cell bead and a singlebarcode bead. In some cases, at least 80%, at least 90%, at least 95%,at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, or atleast 99.99% of the droplets may contain a single cell bead. Moreover,while the first liquid phase and second liquid phase are partitionedinto droplets in this example, other types of partitions can beimplemented at operation 730, including those described elsewhereherein, such as a well.

In operation 740, the barcode can be used to barcode one or moremacromolecular constituents of a given cell bead in a given droplet. Insome cases, the macromolecular constituents of the cell bead aresubjected to conditions sufficient for nucleic acid amplification forbarcoding. In such cases, a barcode can function as a primer in suchamplification. In other cases, ligation can be used for barcoding. Insome cases, the macromolecular constituents are released from the cellbead prior to amplification. In some cases, the barcode is used toidentify one or more macromolecular constituents of the cell bead. Insome cases, a barcoded macromolecule is subjected to nucleic acidsequencing to identify one or more macromolecular components. In somecases, the sequencing is untargeted sequencing. In some cases, thesequencing is targeted sequencing. In some cases, droplets comprise anagent that can release the macromolecular constituents from the cellbead during or prior to barcoding. In some cases, a given barcodedsequencing read can be used to identify the cell (which may have beenencapsulated in a cell bead) from which the barcoded sequencing read wasgenerated. Such capability can link particular sequences to particularcells.

In operation 750, the barcoded macromolecules (or derivatives thereof)can be subjected to sequencing to generate reads. The sequencing may beperformed within a droplet (or partition). The sequencing may beperformed outside of a droplet. For instance, the sequencing may beperformed by releasing the barcoded macromolecules from a droplet (e.g.,by breaking an emulsion comprising the droplets) and sequencing thebarcoded macromolecules using a sequencer, such as an Illumina sequenceror any other sequencer described herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

In some cases, the sequencing is nucleic acid sequencing. In some cases,the nucleic acid sequencing is massively parallel sequencing. In somecases, the nucleic acid sequencing is digital polymerase chain reaction(PCR) sequencing. The sequencing may produce target specific reads frommacromolecular constituents of interest from a cell bead and non-targetspecific reads of other macromolecular sequences. The target specificreads may correspond to one or more nucleic acid sequences from a cellbead. In some cases, the non-target specific reads may arise frommacromolecules external to the cell bead. For instance, the non-targetspecific reads may correspond to one or more exogenous nucleic acidsequences. As another example, the non-target specific reads may arisefrom no-template control effects. The reads may be characterized by atarget specific read to non-target specific read ratio. The targetspecific read to non-target specific read ratio may be greater than 5,greater than 10, greater than 100, greater than 1,000, greater than10,000, greater than greater than 1,000,000, greater than greater than10,000,000, greater than 100,000,000, or greater than 1,000,000,000.

FIG. 8 shows a droplet containing a cell bead and a barcode beadproduced using the method 700. A droplet 800 of aqueous liquid is formedinside a volume 805 of a liquid that is immiscible with the aqueousliquid. The droplet contains a barcode bead 820. The droplet alsocontains a cell bead 810 having an outer surface 830 c and containingone or more macromolecular constituents 815.

FIG. 9 shows a flowchart depicting an example method 900 of producingdroplets containing a cell and a barcode bead (e.g., gel bead)comprising a barcode sequence and generating sequence reads frommacromolecular components of the cell using the PHASE amplificationtechnique described elsewhere herein. In some cases, the method 900comprises the following operations.

In operation 910, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium.

In operation 920, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.The second liquid phase may comprise a cellular growth medium. Thesecond liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of cells. In some cases, the first liquidphase and the second liquid phase are the same phase. In some cases, thefirst liquid phase and the second liquid phase are mixed to provide amixed phase.

In operation 930, the first liquid phase and the second liquid phase canbe brought together with a third liquid phase that is immiscible withthe first and second liquid phase. The third liquid phase may interactwith the first and second liquid phases in such a manner as to partitioneach of the plurality of cells and the plurality of barcode beads into aplurality of droplets. The third liquid phase may comprise an oil andmay also comprise a surfactant. The third liquid phase may comprise afluorinated hydrocarbon. In some cases, a given droplet may include asingle cell and a single barcode bead. In some cases, at least 80%, atleast 90%, at least 95%, at least 99%, at least 99.5%, at least 99.9%,at least 99.95%, or at least 99.99% of the droplets may contain a singlecell. In operation 930, the first liquid phase and the second liquidphase are brought together with a third liquid phase that is immisciblewith the first and second liquid phase. The third liquid phase mayinteract with the first and second liquid phases in such a manner as topartition each of the plurality of cells and the plurality of barcodebeads into a plurality of droplets. The third liquid phase may comprisean oil. The third liquid phase may comprise a fluorinated hydrocarbon.In some cases, a given droplet may include a single cell and a singlebarcode bead. In some cases, at least 80%, at least 90%, at least 95%,at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, or atleast 99.99% of the droplets may contain a single cell. Moreover, whilethe first liquid phase and second liquid phase are partitioned intodroplets in this example, other types of partitions can be implementedat operation 930, including those described elsewhere herein, such as awell.

In operation 940, the cell can be subject to lysis. Lysis may becompleted as described elsewhere herein, including with a lysis agent. Alysis agent may be included within a droplet such that lysis occurswithin the droplet. Lysis of the cell within the droplet can releasemacromolecular constituents from the cell for additional processing,such as barcoding.

In operation 950, the barcode can be used to barcode one or moremacromolecular constituents of a given cell in a given droplet.Barcoding can be completed via PHASE amplification as describedelsewhere herein. Barcode beads can comprise oligonucleotides having abarcode sequence and a primer sequence that hybridizes withmacromolecular constituents released from cells. These oligonucleotidesmay be released from barcode beads, including within droplets. In somecases, the cell is subjected to conditions sufficient for nucleic acidamplification. In some cases, the barcode is used to identify one ormore macromolecular constituents of the cell. In some cases, the barcodeis subjected to nucleic acid sequencing to identify one or moremacromolecular components. In some cases, the sequencing is untargetedsequencing. In some cases, the sequencing is targeted sequencing.

In operation 960, the barcoded macromolecules (or derivatives thereof)can be subjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell from which the barcodedsequencing read was generated. Such capability can link particularsequences to particular cells. Additional details and examples regardingnucleic acid sequencing methods and the use of barcodes foridentification are described elsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 10 shows a flowchart depicting an example method 1000 of producingdroplets containing a cell and a barcode bead (e.g., gel bead)comprising a barcode sequence and generating sequence reads frommacromolecular components of the cell using thedegenerate-oligonucleotide-primed PCR (DOP-PCR) amplification techniquedescribed elsewhere herein. In some cases, the method 1000 comprises thefollowing operations.

In operation 1010, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium.

In operation 1020, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.The second liquid phase may comprise a cellular growth medium. Thesecond liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of cells. In some cases, the first liquidphase and the second liquid phase are the same phase. In some cases, thefirst liquid phase and the second liquid phase are mixed to provide amixed phase.

In operation 1030, the first liquid phase and the second liquid phasecan be brought together with a third liquid phase that is immisciblewith the first and second liquid phase. The third liquid phase mayinteract with the first and second liquid phases in such a manner as topartition each of the plurality of cells and the plurality of barcodebeads into a plurality of droplets. The third liquid phase may comprisean oil and may also comprise a surfactant. The third liquid phase maycomprise a fluorinated hydrocarbon. In some cases, a given droplet mayinclude a single cell and a single barcode bead. In some cases, at least80%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least99.9%, at least 99.95%, or at least 99.99% of the droplets may contain asingle cell. In operation 1030, the first liquid phase and the secondliquid phase are brought together with a third liquid phase that isimmiscible with the first and second liquid phase. The third liquidphase may interact with the first and second liquid phases in such amanner as to partition each of the plurality of cells and the pluralityof barcode beads into a plurality of droplets. The third liquid phasemay comprise an oil. The third liquid phase may comprise a fluorinatedhydrocarbon. In some cases, a given droplet may include a single celland a single barcode bead. In some cases, at least 80%, at least 90%, atleast 95%, at least 99%, at least 99.5%, at least 99.9%, at least99.95%, or at least 99.99% of the droplets may contain a single cell.Moreover, while the cells are partitioned into droplets in this example,other types of partitions can be implemented at operation 1030,including those described elsewhere herein, such as a well.

In operation 1040, the cell can be subjected to lysis. Lysis may becompleted as described elsewhere herein, including with a lysis agent. Alysis agent may be included within a droplet such that lysis occurswithin the droplet. Lysis of the cell within the droplet can releasemacromolecular constituents from the cell for additional process, suchas barcoding.

In operation 1050, the barcode can be used to barcode one or moremacromolecular constituents of a given cell in a given droplet.Barcoding can be completed via DOP-PCR amplification. Barcode beads cancomprise oligonucleotides having a barcode sequence and a primersequence that hybridizes with macromolecular constituents released fromcells. These oligonucleotides may be released from beads, includingwithin droplets. In some cases, the macromolecular constituents of thecell are subjected to conditions sufficient for nucleic acidamplification. In some cases, the barcode is used to identify one ormore macromolecular constituents of the cell. In some cases, the barcodeis subjected to nucleic acid sequencing to identify one or moremacromolecular components. In some cases, the sequencing is untargetedsequencing. In some cases, the sequencing is targeted sequencing.

In operation 1060, the barcoded macromolecules (or derivatives thereof)can be subjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell from which the barcodedsequencing read was generated. Such capability can link particularsequences to particular cells. Additional details and examples regardingnucleic acid sequencing methods and the use of barcodes foridentification are described elsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 11 shows a flowchart depicting an example method 1100 of producingdroplets containing a cell bead and a barcode bead (e.g., a gel bead)comprising a barcode sequence and generating sequence reads frommacromolecular components of a the cell bead. The cell bead is generatedby cross-linking of at least a portion of a cell. In some cases, themethod 1100 may comprise the following operations.

In operation 1110, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium.

In operation 1120, the cells can be subjected to conditions sufficientto cross-link at least a portion of the cells. In some cases, the cellsare subjected to conditions sufficient to cross-link at least a portionof a membrane. In some cases, the cells are subjected to conditionssufficient to cross-link the entirety of a membrane. The cross-linkingmay be achieved by exposing the cells todiothiobis(succinimidylpropionate) (DSP). The cross-linking may beachieved by exposing the cells to any cross-linking agent. Thecross-linked portion of the cells may be diffusively permeable tochemical or biochemical reagents. The cross-linked portion may bediffusively impermeable to macromolecular constituents of the cells. Inthis manner, the cross-linked portion may act to allow the cells to besubjected to chemical or biochemical operations while spatiallyconfining the macromolecular constituents to a region of the dropletdefined by the cross-linked portion.

In operation 1130, the cross-linked cells can be subjected to conditionssufficient to lyse the cross-linked cells. In some cases, lysis may becompleted in a droplet, such as, for example, via a lysis agent in adroplet. The lysis of the cross-linked cells may occur subsequent tosubjecting the cross-linked cells to conditions sufficient to cross-linkthe cells. In some cases, the lysis of the cross-linked cells may occurcontemporaneously with subjecting the cells to conditions sufficient tocross-link the cells. In some cases, lysis may be completed in bulk withmultiple cross-linked cells treated in one pot. The lysis may disruptcomponents of the cross-linked cell that aid in containingmacromolecular constituents of the cells. However, the cross-linking ofthe cell may provide a barrier such that the “released” materials arestill retained within the cross-linked cell. The lysis may be achievedby exposing the cross-linked cells to sodium hydroxide (NaOH), potassiumhydroxide (KOH), or any other alkaline agent. The lysis may be achievedby exposing the cross-linked cells to a detergent, such as sodiumdodecyl sulfate (SDS), 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethyleneglycol (TRITON X-100) or any non-ionic surfactant, or a saponin. Thelysis may be achieved by exposing the cross-linked cells to an enzyme,such as a proteinase or a lytic enzyme (such as a lysozyme, cellulose,or zymolase). The lysis may be achieved by exposing the cross-linkedcells to freeze thawing. The lysis may be achieved by exposing thecross-linked cells to electromagnetic radiation, such as ultraviolet(UV) light. The lysis may be achieved by exposing the cross-linked cellsto heat. The lysis may be achieved by exposing the cross-linked cells toany other lysis agent.

In operation 1140, the lysed, cross-linked cells can be subjected toconditions sufficient to denature one or more macromolecularconstituents of the lysed, cross-linked cells. In some cases,denaturation is achieved in bulk, where more than one cross-linked cellis subjected to denaturation conditions in a single pot. The denaturingmay be achieved by exposing the cross-linked cells to sodium hydroxide(NaOH). The denaturing may be achieved by exposing the cross-linkedcells to any other denaturing agent. In some examples, operation 1140 iscompleted contemporaneous to operation 1130. In some examples, adenaturing agent can both denature macromolecular constituents and lysethe cross-linked cells.

In operation 1150, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.The second liquid phase may comprise a cellular growth medium. Thesecond liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of cross-linked cells. In some cases, thefirst liquid phase and the second liquid phase are the same phase. Insome cases, the first liquid phase and the second liquid phase are mixedto provide a mixed phase.

In operation 1160, the first liquid phase and the second liquid phasecan be brought together with a third liquid phase that is immisciblewith the first and second liquid phase. The third liquid phase mayinteract with the first and second liquid phases in such a manner as topartition each of the plurality of cross-linked cells and the pluralityof barcode beads into a plurality of droplets. The third liquid phasemay comprise an oil and may also comprise a surfactant. The third liquidphase may comprise a fluorinated hydrocarbon. In some cases, a givendroplet may include a single cross-linked cell and a single barcodebead. In some cases, at least 80%, at least 90%, at least 95%, at least99%, at least 99.5%, at least 99.9%, at least 99.95%, or at least 99.99%of the droplets may contain a single cross-linked cell. Moreover, whilecross-linked cells are partitioned into droplets in this example, othertypes of partitions can be implemented at operation 1160, includingthose described elsewhere herein, such as a well.

In operation 1170, the cross-linked cells can be subjected to conditionssufficient to reverse the cross-linking. The reversal of thecross-linking may be achieved by exposing the cross-linked cells to areducing agent (e.g., dithiothreitol (DTT)), which may be present in adroplet. The reversal of the cross-linking may be achieved by exposingthe cross-linked cells to any substance capable of reversingcross-linking. Reversal of cross-linking can release the macromolecularconstituents of the cross-linked cells to the interiors of the droplets.In some cases, operation 1170 also includes releasing barcodes from thebarcode beads, which may be achieved with the same stimulus, such as,for example used to reverse cross-linking of the cells. In some cases,the stimuli are different. Released barcodes can then participate inbarcoding as in operation 1180.

In operation 1180, the barcode can be used to barcode one or moremacromolecular constituents of a given cross-linked cell in a givendroplet. In some cases, the macromolecular constituents are subjected toconditions sufficient for nucleic acid amplification for barcoding. Insuch cases, the barcodes released from the barcode beads can function asprimers in such amplification. In some cases, ligation is used forbarcoding. In some cases, the barcode is used to identify one or moremacromolecular constituents of the cross-linked cell. In some cases, thebarcode is subjected to nucleic acid sequencing to identify one or moremacromolecular components. In some cases, the sequencing is untargetedsequencing. In some cases, the sequencing is targeted sequencing.

In operation 1190, the barcoded macromolecules (or derivatives thereof)can be subjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell (which may have been across-linked cell) from which the barcoded sequencing read wasgenerated. Such capability can link particular sequences to particularcells. Additional details and examples regarding nucleic acid sequencingmethods and the use of barcodes for identification are describedelsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 12 shows a droplet containing a cross-linked cell and a barcodebead produced using the method 1100. A droplet 1200 of aqueous liquid isformed inside a volume 1205 of a liquid that is immiscible with theaqueous liquid. The droplet contains a single gel bead 1220. The dropletalso contains a single cross-linked cell 1210 containing one or moremacromolecular constituents 1215. A portion of the cross-linked cell iscrosslinked to form a crosslinked outer portion 1230 c.

FIG. 13 shows a flowchart that depicts an example method 1300 ofproducing droplets containing a cell bead (e.g., comprising a cell orcomponents of a cell) and a barcode bead (e.g., gel bead) comprisingbarcode sequences and generating sequence reads from macromolecularcomponents of a cell of which cell or components have been encapsulatedby a polymer or gel. In some cases, the method 1300 may comprise thefollowing operations.

In operation 1310, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium. The first liquid phase may furthercomprise precursors that are capable of being polymerized or gelled. Theprecursors that are capable of being polymerized or gelled may comprisepoly(acrylamide-co-acrylic acid). The first liquid phase may furthercomprise a first agent that is completely or partially capable ofpolymerizing or gelling the precursors, such as an acylating agent. Theacylating agent may comprise4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The first liquid phase may comprise other salts of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Additionalexamples of precursors include polyacrylamide, species comprising adisulfide bond (e.g., cystamine (2,2′-dithiobis(ethylamine), disulfidecross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol,polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol,PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid,collagen, fibrin, gelatin, or elastin. Moreover, in some cases,precursors are pre-formed polymer chains that can be crosslinked (e.g.,via gelation) to form larger structures such as beads. In some cases,precursors may be monomeric species that are polymerized to form largerstructures such as beads.

The first liquid phase may further comprise one or more of a magneticparticle, reagents for reverse transcription (e.g., oligonucleotideprimers or reverse transcriptase), reagents for nucleic acidamplification (e.g., primers (e.g. random primers, primers specific forgiven DNA loci), polymerases, nucleotides (e.g. unmodified nucleotides,modified nucleotides, or non-canonical nucleotides), co-factors (e.g.,ionic co-factors)) or reagents for nucleic acid modification, includingligation, digestion, methylation, random mutagenesis, bisulfiteconversion, uracil hydrolysis, nucleic acid repair, nucleic acidinsertion or cleavage (e.g. via CRISPR/Cas9-mediated ortransposon-mediated insertion or cleavage), capping and decapping.

In operation 1320, the first liquid phase can be brought into contactwith an immiscible second liquid phase to form a plurality of droplets.The third liquid phase may comprise an oil and may also comprise asurfactant. The third liquid phase may comprise a fluorinatedhydrocarbon. In some cases, a given droplet may include a single celland precursors that are capable of being polymerized or gelled. In somecases, at least 80%, at least 90%, at least 95%, at least 99%, at least99.5%, at least 99.9%, at least 99.95%, or at least 99.99% of thedroplets may contain a single cell.

In operation 1330, the droplets can be subjected to conditionssufficient to polymerize or gel the precursors. The conditionssufficient to polymerize or gel the precursors may comprise exposure toheating, cooling, electromagnetic radiation, or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around thecells or cell components, such that they are encapsulated in cell beads.The polymer or gel may be diffusively permeable to chemical orbiochemical reagents. The polymer or gel may be diffusively impermeableto macromolecular constituents of the cells or cell components. In thismanner, the polymer or gel may act to allow the cell beads to besubjected to chemical or biochemical operations while spatiallyconfining the contents of the cells beads to a region defined by thepolymer or gel.

The cell beads may be functionalized to bind to targeted analytes, suchas nucleic acids, proteins, or other analytes. The polymer or gel of thecell beads may be polymerized or gelled via a passive mechanism. Thepolymer or gel may be stable in alkaline conditions or at elevatedtemperature. The polymer or gel may have mechanical properties similarto the mechanical properties (e.g., tensile strength) of a bead. Thepolymer or gel may be of a lower density than an oil. The cell beads maybe of a density that is roughly similar to that of a buffer. The cellbeads may have a tunable pore size. The pore size may be chosen to, forinstance, retain denatured nucleic acids. The pore size may be chosen tomaintain diffusive permeability to exogenous chemicals such as sodiumhydroxide (NaOH) and/or endogenous chemicals such as inhibitors. Thecell beads may be biocompatible. The polymer or gel of the cell beadsmay maintain or enhance cell viability. The cell beads may bebiochemically compatible. The polymer or gel of the cell beads may bepolymerized and/or depolymerized thermally, chemically, enzymatically,and/or optically.

In some examples, the resulting cell beads may comprisepoly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages.The preparation of these cell beads may comprise a two-operationreaction. In the first activation operation, poly(acrylamide-co-acrylicacid) may be exposed to an acylating agent to convert carboxylic acidsto esters. For instance, the poly(acrylamide-co-acrylic acid) may beexposed to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride (DMTMM). The polyacrylamide-co-acrylic acid may be exposed toother salts of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. In the secondcross-linking operation, the ester formed in the first operation may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twooperations, an encapsulated cell or its components are surrounded bypolymeric strands, such as polyacrylamide strands linked together bydisulfide bridges thereby resulting in a cell bead. In this manner, thecell may be encased inside of the cell bead. In some cases, one or moremagnetic (e.g., paramagnetic) particles may be encapsulated within acell bead such, as for example, by also including such particles withina droplet along with polymeric precursors.

Cell beads may be or include a cell, cell derivative, cellular materialand/or material derived from the cell in, within, or encased in amatrix, such as a polymeric matrix. A cell encapsulated by a bead may bea live cell.

In operation 1340, cell beads generated from precursors in droplets aresuspended in the second liquid phase may be resuspended into an aqueousenvironment by a solvent exchange process. Such processing can promotethe processing of cell beads with additional aqueous phase materials.The solvent exchange process may comprise the operations of collectingcell beads in droplets (for instance, in an Eppendorf tube or othercollection vessel), removing excess oil (for instance, by pipetting),adding a ligation buffer (such as a 3× ligation buffer), vortexing,adding a buffer (such as a 1× 1H,1H,2H,2H-perfluoro-1-octanol (PFO)buffer), vortexing, centrifugation, and separation. The separationoperation may comprise magnetic separation via attraction ofencapsulated magnetic particles. The magnetic separation may beaccomplished by using a magnetic separating apparatus to pull cell beadscontaining magnetic particles away from unwanted remaining oil andsolvents. For instance, the magnetic separation apparatus may be used topull cell beads containing magnetic particles away from the ligationbuffer and PFO to allow removal of the ligation buffer and PFO (forinstance by pipetting). The cell beads containing magnetic particles maythen be suspended in a ligation buffer and vortexed. The cell beadscontaining paramagnetic particles may again be separated magneticallyand the ligation buffer may be removed. This cycle of re-suspension,vortexing, and magnetic separation may be repeated until the cell beadsare free or substantially free of oil phase and suspended in aqueousmedium. For instance, the cycle may be repeated 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10 times. The cell beads may then be processed inaqueous phases and with additional materials.

Once the cell beads are in an aqueous medium, the cell beads may befurther treated. For instance, the cell beads in aqueous solution may befiltered (for instance, using a 70 μm filter) to remove clumps and/orlarge cell beads from the solution. In some cases, additional reagentsmay be added to and/or removed from the aqueous medium to furtherprocess the cell beads. Further processing can include, withoutlimitation, reverse transcription, nucleic acid amplification, andnucleic acid modification of macromolecular constituents within the cellbeads.

In operation 1350, the cell beads can be subjected to conditionssufficient to lyse the cells encapsulated in the cell beads. In somecases, lysis is completed via a lysis agent present in a droplet. Insome cases, lysis is completed in bulk, for example with the aid of alysis agent that contacts a plurality of cell beads in one pot. In somecases, the lysis of the cells occurs subsequent to subjecting the cellsto conditions sufficient to encapsulate the cells in the polymer or gel.The lysis may release macromolecular constituents of the lysed cells.The lysis may be achieved by exposing the cell beads to sodium hydroxide(NaOH), potassium hydroxide (KOH), or any other alkaline agent. Thelysis may be achieved by exposing the cell beads to a detergent, such assodium dodecyl sulfate (SDS),4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100) orany non-ionic surfactant, or a saponin. The lysis may be achieved byexposing the cell beads to an enzyme, such as a proteinase or a lyticenzyme (such as a lysozyme, cellulose, or zymolase). The lysis may beachieved by exposing the cell beads to freeze thawing. The lysis may beachieved by exposing the cell beads to electromagnetic radiation, suchas ultraviolet (UV) light. The lysis may be achieved by exposing thecell beads to heat. The lysis may be achieved by exposing the cell beadsto any other lysis agent. A cell bead may retain species released fromlysed cells within the cell bead, such as, for example, via itspolymeric or gel structure.

In operation 1360, the cell beads can be subjected to conditionssufficient to denature one or more macromolecular constituents releasedby the lysed cells. In some cases, denaturation occurs in bulk wheremore than one cell bead is subjected to denaturation conditions in asingle pot. In some cases, denaturation is achieved via a denaturationagent present in a droplet. The denaturing may be achieved by exposingthe cell beads to sodium hydroxide (NaOH). The denaturing may beachieved by exposing the cell beads to any other denaturing agent. Insome cases, operation 1360 is completed contemporaneously with operation1350. In some examples, a denaturing agent can both denaturemacromolecular constituents and lyse the cells within the cell beads.

In operation 1370, a fourth liquid phase comprising a plurality ofbarcode beads can be provided. The fourth liquid phase may be aqueous.The fourth liquid phase may comprise a cellular growth medium. Thefourth liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of cell beads. In some cases, the thirdliquid phase and the fourth liquid phase are the same phase. In somecases, the third liquid phase and the fourth liquid phase are mixed toprovide a mixed phase.

In operation 1380, the third liquid phase and the fourth liquid phasecan be brought together with a fifth liquid phase that is immisciblewith the third and fourth liquid phases. The fifth liquid phase mayinteract with the third and fourth liquid phases in such a manner as topartition cells beads encapsulating cellular material and the pluralityof barcode beads into a plurality of droplets. The fifth liquid phasemay comprise an oil and may also comprise a surfactant. The fifth liquidphase may comprise a fluorinated hydrocarbon. In some cases, a givendroplet may include a single cell bead and a single barcode bead. Insome cases, at least 80%, at least 90%, at least 95%, at least 99%, atleast 99.5%, at least 99.9%, at least 99.95%, or at least 99.99% of thedroplets may contain a single cell bead. Moreover, while the cell beadsand barcode beads are partitioned into droplets in this example, othertypes of partitions can be implemented in operation 1380, includingthose described elsewhere herein, such as a well.

In operation 1390, the cell beads are subjected to conditions sufficientto release the macromolecular constituents from cell beads. The releaseof the macromolecular constituents may be achieved by exposing cellbeads to a reducing agent (e.g., dithiothreitol (DTT)), which may bepresent in a droplet. The release of the macromolecular constituents maybe achieved by exposing the cell beads to any substance capable ofreleasing the macromolecular constituents. In some cases, operation 1390also includes releasing barcodes from the barcode beads, which may beachieved with the same stimulus, such as, for example, that used torelease macromolecular constituents from cell beads. In some cases, thestimuli are different. Released barcodes can then participate inbarcoding as in operation 1392.

In operation 1392, the barcode is used to barcode one or moremacromolecular constituents of a given cell bead in a given droplet. Insome cases, the macromolecular constituents of the cell bead aresubjected to conditions sufficient for nucleic acid amplification forbarcoding. In such cases, the barcode may function as a primer duringsuch amplification. In other cases, ligation can be used for barcoding.In some cases, the barcode is used to identify one or moremacromolecular constituents of the cell bead. In some cases, the barcodeis subjected to nucleic acid sequencing to identify one or moremacromolecular components. In some cases, the sequencing is untargetedsequencing. In some cases, the sequencing is targeted sequencing.

In operation 1394, barcoded macromolecules (or derivatives thereof) aresubjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell (which may have beenencapsulated in a cell bead) from which the barcoded sequencing read wasgenerated. Such capability can link particular sequences to particularcells. Additional details and examples regarding nucleic acid sequencingmethods are described elsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 14 shows a droplet containing a single cell bead and a singlebarcode bead produced using the method 1300. A droplet 1400 of aqueousliquid is formed inside a volume 1405 of a liquid that is immisciblewith the aqueous liquid. The droplet contains a single barcode bead1420. The droplet also contains a cell 1410 containing one or moremacromolecular constituents 1415. The cell may be surrounded by a gel orpolymer 1430 d and is encapsulated within a cell bead 1430 d.

FIG. 25 shows a flowchart that depicts an example method 2500 ofproducing droplets containing a cell bead comprising a cell and abarcode bead (e.g., gel bead) comprising barcode sequences andgenerating sequence reads from macromolecular components of the cell. Insome cases, the method 2500 comprises the following operations.

In operation 2510, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium. The first liquid phase may furthercomprise precursors that are capable of being polymerized or gelled. Theprecursors that are capable of being polymerized or gelled may comprisepoly(acrylamide-co-acrylic acid). The first liquid phase may furthercomprise a first agent that is completely or partially capable ofpolymerizing or gelling the precursors, such as an acylating agent. Theacylating agent may comprise4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The first liquid phase may comprise other salts of4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Additionalexamples of precursors include polyacrylamide, species comprising adisulfide bond (e.g., cystamine (2,2′-dithiobis(ethylamine), disulfidecross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol,polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol,PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid,collagen, fibrin, gelatin, or elastin. Moreover, in some cases,precursors are pre-formed polymer chains that can be crosslinked (e.g.,via gelation) to form larger structures such as beads. In some cases,precursors may be monomeric species that are polymerized to form largerstructures such as beads.

The first liquid phase may further comprise one or more of a magneticparticle, reagents for reverse transcription (e.g., oligonucleotideprimers or reverse transcriptase), reagents for nucleic acidamplification (e.g., primers (e.g. random primers, primers specific forgiven DNA loci), polymerases, nucleotides (e.g. unmodified nucleotides,modified nucleotides, or non-canonical nucleotides), co-factors (e.g.,ionic co-factors)) or reagents for nucleic acid modification, includingligation, digestion, methylation, random mutagenesis, bisulfiteconversion, uracil hydrolysis, nucleic acid repair, nucleic acidinsertion or cleavage (e.g. via CRISPR/Cas9-mediated ortransposon-mediated insertion or cleavage), capping and decapping.

In operation 2520, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.The second liquid phase may comprise a cellular growth medium. Thesecond liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of cell beads. In some cases, the firstliquid phase and the third liquid phase are the same phase. In somecases, the first liquid phase and the second liquid phase are mixed toprovide a mixed phase.

In operation 2530, the first liquid phase and second liquid phase can bebrought together, if not already mixed, and two are brought into contactwith an immiscible second liquid phase to form a plurality of droplets.The third liquid phase may comprise an oil and may also comprise asurfactant. The third liquid phase may comprise a fluorinatedhydrocarbon. In some cases, a given droplet may include a single celland precursors that are capable of being polymerized or gelled. In somecases, at least 80%, at least 90%, at least 95%, at least 99%, at least99.5%, at least 99.9%, at least 99.95%, or at least 99.99% of thedroplets may contain a single cell.

In operation 2540, the droplets are subjected to conditions sufficientto polymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, or light. The conditions sufficientto polymerize or gel the precursors may comprise any conditionssufficient to polymerize or gel the precursors. Following polymerizationor gelling, a polymer or gel may be formed around the cells and barcodebeads, such that the cells and barcode beads are encapsulated in cellbeads. The polymer or gel of the cell beads may be diffusively permeableto chemical or biochemical reagents. The polymer or gel of the cellbeads may be diffusively impermeable to macromolecular constituents ofthe cells. In this manner, the polymer or gel may act to allow the cellsto be subjected to chemical or biochemical operations while spatiallyconfining the macromolecular constituents to a region of the dropletdefined by the polymer or gel.

The polymer or gel of the cell beads may be functionalized to bind totargeted analytes, such as nucleic acids, proteins, or other analytes.The polymer or gel of the cell beads may be polymerized or gelled via apassive mechanism. The polymer or gel of the cell beads may be stable inalkaline conditions or at elevated temperature. The polymer or gel ofthe cell beads may be of a lower density than an oil. The polymer or gelof the cell beads may be of a density that is roughly similar to that ofa buffer. The polymer or gel of the cell beads may have a tunable poresize. The pore size may be chosen to, for instance, retain denaturednucleic acids. The pore size may be chosen to maintain diffusivepermeability to exogenous chemicals such as sodium hydroxide (NaOH)and/or endogenous chemicals such as inhibitors. The polymer or gel ofthe cell beads may be biocompatible. The polymer or gel of the cellbeads may maintain or enhance cell viability. The polymer or gel of thecell beads may be biochemically compatible. The polymer or gel of thecell beads may be polymerized and/or depolymerized thermally,chemically, enzymatically, and/or optically.

In some examples, the resulting cell beads may comprisepoly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages.The preparation of this polymer may comprise a two-operation reaction.In the first activation operation, poly(acrylamide-co-acrylic acid) maybe exposed to an acylating agent to convert carboxylic acids to esters.For instance, the poly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking operation, the ester formed in the firstoperation may be exposed to a disulfide crosslinking agent. Forinstance, the ester may be exposed to cystamine(2,2′-dithiobis(ethylamine)). Following the two operations, anencapsulated cell and barcode bead is surrounded by polymeric strands,such as polyacrylamide strands linked together by disulfide bridgesthereby resulting in a cell bead comprising the cell and barcode bead.In this manner, the cell and barcode bead may be encased inside of thecell bead. In some cases, one or more magnetic (e.g., paramagnetic)particles may be encapsulated within the cell bead such, as for example,by also including such particles within a droplet along with polymericprecursors.

Cell beads may be or include a cell, cell derivative, cellular materialand/or material derived from the cell in, within, or encased in amatrix, such as a polymeric matrix. A cell encapsulated by a bead may bea live cell.

In operation 2550, cell beads generated from precursors, cells andbarcode beads in droplets are suspended in the third liquid phase andmay be resuspended into a fourth liquid phase (e.g., an aqueous phase)by a solvent exchange process. Such processing can promote theprocessing of cell beads with additional aqueous phase materials. Thesolvent exchange process may comprise the operations of collecting cellbeads in droplets (for instance, in an Eppendorf tube or othercollection vessel), removing excess oil (for instance, by pipetting),adding a ligation buffer (such as a 3× ligation buffer), vortexing,adding a buffer (such as a 1× 1H,1H,2H,2H-perfluoro-1-octanol (PFO)buffer), vortexing, centrifugation, and separation. The separationoperation may comprise magnetic separation via attraction ofencapsulated magnetic particles. The magnetic separation may beaccomplished by using a magnetic separating apparatus to pull cell beadscontaining magnetic particles away from unwanted remaining oil andsolvents. For instance, the magnetic separation apparatus may be used topull cell beads containing magnetic particles away from the ligationbuffer and PFO to allow removal of the ligation buffer and PFO (forinstance by pipetting). The cell beads containing magnetic particles maythen be suspended in a ligation buffer and vortexed. The cell beadscontaining paramagnetic particles may again be separated magneticallyand the ligation buffer may be removed. This cycle of re-suspension,vortexing, and magnetic separation may be repeated until the cell beadsare free or substantially free of oil phase and suspended in aqueousmedium. For instance, the cycle may be repeated 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10 times. The cell beads may then be processed inaqueous phases and with additional materials in aqueous phases.

Once the cell beads are in an aqueous medium, the cell beads may befurther treated. For instance, the cell beads in aqueous solution may befiltered (for instance, using a 70 μm filter) to remove clumps and/orlarge cell beads from the solution. In some cases, additional reagentsmay be added to and/or removed from the aqueous medium to furtherprocess the cell beads. Further processing can include, withoutlimitation, reverse transcription, nucleic acid amplification, andnucleic acid modification of macromolecular constituents within the cellbeads.

In operation 2560, the cell beads can be subjected to conditionssufficient to lyse the cells encapsulated in the cell beads. In somecases, lysis is completed via a lysis agent present in a droplet. Insome cases, lysis is completed in bulk, for example with the aid of alysis agent that contacts a plurality of cell beads in one pot. In somecases, the lysis of cells of the cell beads occurs subsequent tosubjecting the cells to conditions sufficient to encapsulate the cellsin the polymer or gel. The lysis may release macromolecular constituentsof the lysed cells of the cell beads. The lysis may be achieved byexposing the cell beads to sodium hydroxide (NaOH), potassium hydroxide(KOH), or any other alkaline agent. The lysis may be achieved byexposing the cell beads to a detergent, such as sodium dodecyl sulfate(SDS), 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITONX-100) or any non-ionic surfactant, or a saponin. The lysis may beachieved by exposing the cell beads to an enzyme, such as a proteinaseor a lytic enzyme (such as a lysozyme, cellulose, or zymolase). Thelysis may be achieved by exposing the cell beads to freeze thawing. Thelysis may be achieved by exposing the cell beads to electromagneticradiation, such as ultraviolet (UV) light. The lysis may be achieved byexposing the cell beads to heat. The lysis may be achieved by exposingthe cell beads to any other lysis agent. A cell bead may retain speciesreleased from lysed cells within the cell bead, such as, for example,via its polymeric or gel structure.

In operation 2570, the cell beads can be subjected to conditionssufficient to denature one or more macromolecular constituents releasedby the lysed cells within the cell beads. In some cases, denaturationoccurs in bulk where more than one cell bead is subjected todenaturation conditions in a single pot. In some cases, denaturation isachieved via a denaturation agent present in a droplet. The denaturingmay be achieved by exposing the cell beads to sodium hydroxide (NaOH).The denaturing may be achieved by exposing the cell beads to any otherdenaturing agent. In some cases, operation 2570 is completedcontemporaneously with operation 2560. In some examples, a denaturingagent can both denature macromolecular constituents and lyse the cellsof the cell beads.

In operation 2580, the fourth liquid phase, having the cell beads, isbrought into contact with a fifth liquid phase that is immiscible withthe fourth liquid phase. The fifth liquid phase may interact with thefourth liquid phase in such a manner as to partition cell beads into aplurality of droplets. The fifth liquid phase may comprise an oil andmay also comprise a surfactant. The fifth liquid phase may comprise afluorinated hydrocarbon. In some cases, a given droplet may include asingle cell bead. In some cases, at least 80%, at least 90%, at least95%, at least 99%, at least 99.5%, at least 99.9%, at least 99.95%, orat least 99.99% of the droplets may contain a single cell bead. In somecases, additional precursors are added to the fourth liquid phase,droplets generated and the precursors polymerized or gelled (includingas described herein) to generate even larger cell beads comprising thecell bead. The larger cell beads can be stored for future use. Moreover,while the cell beads are partitioned into droplets in this example,other types of partitions can be implemented at operation 2580,including those described elsewhere herein, such as a well.

In operation 2590, the cell beads can then be subjected to conditionssufficient to release the barcode beads and macromolecular constituentsof cells from the cell beads. The release of the macromolecularconstituents may be achieved by exposing the cell beads to a reducingagent (e.g., dithiothreitol (DTT)), which may be present in the droplet.The release of the macromolecular constituents may be achieved byexposing the cell beads to any substance capable of releasing themacromolecular constituents. In some cases, operation 2590 also includesreleasing barcodes from the barcode beads, which may be achieved withthe same stimulus, such as, for example, used to reverse cross-linkingof the cell bead. In some cases, the stimuli are different. Releasedbarcodes can then participate in barcoding as in operation 2592.

In operation 2592, the barcodes is used to barcode one or moremacromolecular constituents of a given cell bead in a given droplet. Insome cases, the macromolecular constituents of the cell bead aresubjected to conditions sufficient for nucleic acid amplification forbarcoding. In such cases, a barcode may function a primer during suchamplification. In some cases, ligation is used for barcoding. In somecases, the barcode is used to identify one or more macromolecularconstituents of the cell bead. In some cases, the barcode is subjectedto nucleic acid sequencing to identify one or more macromolecularcomponents. In some cases, the sequencing is untargeted sequencing. Insome cases, the sequencing is targeted sequencing.

In operation 2594, barcoded macromolecules (or derivatives thereof) aresubjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell from which the barcodedsequencing read was generated. Such capability can link particularsequences to particular cells. Additional details and examples regardingnucleic acid sequencing methods are described elsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 26A shows a droplet 2610 containing a cell bead 2620 thatencapsulates a cell 2630 and a single gel bead 2640 comprising a barcodesequence produced using the method 2500. FIG. 26B shows a larger cellbead 2650 comprising the elements of droplet 2610 in FIG. 26A, where thelarger cell bead 2650 has been generated from precursors present in adroplet and subsequently polymerized or gelled.

FIG. 15 shows a flowchart depicting an example method 1500 of producingdroplets containing a droplet containing a cell bead, a barcode bead(e.g., a gel bead), and generating sequence reads from macromolecularcomponents a cell associated with the cell bead. In some cases, themethod 1500 may comprise the following operations.

In operation 1510, a first liquid phase comprising a plurality of cells,precursors capable of being polymerized or gelled and a denaturant isprovided. The first liquid phase may be aqueous. The first liquid phasemay comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium.

In operation 1520, the first liquid phase can be brought together with asecond liquid phase that is immiscible with the first liquid phase. Thefirst liquid phase may interact with the second liquid phase in such amanner as to partition each of the plurality of cells into a pluralityof first droplets that also include polymer or gel precursors anddenaturant. The second liquid phase may comprise an oil. The secondliquid phase may comprise a fluorinated hydrocarbon. In some cases, agiven first droplet may include a single cell. In some cases, at least80%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least99.9%, at least 99.95%, or at least 99.99% of the first droplets maycontain a single cell.

In operation 1530, the second liquid phase can be brought into contactwith an immiscible third phase comprising a plurality of barcode beadscomprising barcodes and a denaturant neutralization agent. The thirdliquid phase may be aqueous. The barcode beads each contain a barcode tobarcode one or more macromolecular constituents of the plurality ofcells. In some cases, the first liquid phase and the third liquid phaseare the same phase. The bringing together of the second liquid phase andthe third liquid phase can generate a mixture comprising the barcodebeads and the first droplets.

In operation, 1540, the mixture generated in operation 1530 can bebrought into contact with an immiscible fourth liquid phase to formsecond droplets having the first droplets and beads (e.g., a dropletwithin a droplet configuration). The fourth liquid phase may interactwith the mixture in such a manner as to partition each of the firstdroplets and the plurality of barcode beads into a plurality of seconddroplets. The fourth liquid phase may comprise an oil and may alsoinclude a surfactant. The fourth liquid phase may comprise a fluorinatedhydrocarbon. In some cases, a given second droplet may include a singlefirst droplet and a single barcode bead. In some cases, at least 80%, atleast 90%, at least 95%, at least 99%, at least 99.5%, at least 99.9%,at least 99.95%, or at least 99.99% of the second droplets may contain asingle first droplet.

In operation 1550, the cells in the first droplets can be subjected toconditions sufficient to lyse the cells. In some cases, lysis iscompleted with the aid of a lysis agent in a droplet. The lysis mayrelease macromolecular constituents of the lysed cell bead into thefirst droplet. The lysis may be achieved via the action of thedenaturant (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), orany other alkaline agent) also present in the first droplet. In somecases, the lysis may be achieved with a detergent present in the firstdroplet, such as sodium dodecyl sulfate (SDS),4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100) orany non-ionic surfactant, or a saponin. The lysis may be achieved withan enzyme in the first droplet, such as a proteinase or a lytic enzyme(such as a lysozyme, cellulose, or zymolase). The lysis may be achievedby exposing first droplet to freeze thawing. The lysis may be achievedby exposing the first droplet to electromagnetic radiation, such asultraviolet (UV) light. The lysis may be achieved by exposing the firstdroplet to heat. The lysis may be achieved by exposing the contents ofthe first droplet to any other lysis agent.

In operation 1560, the lysed cells can then be subjected to conditionssufficient to denature one or more macromolecular constituents releasedby the lysed cells. In some examples, lysis is completed with the aid ofa denaturation agent present in the first droplet, such as, for examplesodium hydroxide (NaOH). In some cases, the denaturation agent ispresent outside the first droplet. In some examples, the same denaturingagent can both denature macromolecular constituents and lyse the cells.

In operation 1570, the droplets generated in operation 1540 can besubjected to conditions sufficient to polymerize or gel the precursorswithin first droplets. Polymerization or gelling of the precursors cangenerate cell beads that encapsulate released/denatured macromolecularcomponents from the lysed cells. In cases where a first dropletgenerated in operation 1520 comprises a single cell, the resultinggenerated from that droplet will also comprise macromolecularconstituents of the single cell. The conditions sufficient to polymerizeor gel the precursors may comprise exposing the first droplets toheating, cooling, electromagnetic radiation, or light. The conditionssufficient to polymerize or gel the precursors may comprise any exposingthe first droplets conditions sufficient to polymerize or gel theprecursors. Following polymerization or gelling, a polymer or gel may beformed around the material released from cell lysis to generate a cellbead. The cell bead may be diffusively permeable to chemical orbiochemical reagents. The cell bead may be diffusively impermeable tomacromolecular constituents of the cell bead. In this manner, thepolymer or gel may act to allow the cell bead to be subjected tochemical or biochemical operations while spatially confining themacromolecular constituents to a region of the droplet defined by thepolymer or gel. The polymer or gel of the cell bead may include one ormore of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel of thecell bead may comprise any other polymer or gel. In some cases,polymerization of the precursors in the first droplets generates cellbeads comprising the macromolecular constituents of cells and alsoreleases the cells beads from the first droplets and into the interiorsof the second droplets. Upon release of the cell beads from the firstdroplets, denaturant neutralization agent present in interiors of thesecond droplets neutralizes the denaturant that is also released withthe cell beads. Polymerization may also be coupled to or precede asolvent exchange process that aids in releasing cell beads from thefirst droplets and into the interiors of the second droplets.

In operation 1580, the cell beads can be subjected to conditionssufficient to release the macromolecular constituents from cell beads.The release of the macromolecular constituents may be achieved byexposing the cell beads to a reducing agent (e.g., dithiothreitol(DTT)), which may be present in a droplet. The release of themacromolecular constituents may be achieved by exposing the cell beadsto any substance capable of releasing the macromolecular constituents.In some cases, operation 1580 also includes releasing barcodes from thebarcode beads in the second droplets which may be achieved with the samestimulus, such as, for example, used to reverse cross-linking of thecell bead. In some cases, the stimuli are different. Released barcodescan then participate in barcoding as in operation 1590.

In operation 1590, barcodes can be used to barcode one or moremacromolecular constituents of a given single cell bead in a givensecond droplet. In some cases, the macromolecular constituents of thecell bead are subjected to conditions sufficient for nucleic acidamplification for barcoding. In such cases, a barcode can function as aprimer in such amplification. In other cases, ligation may be used forbarcoding. In some cases, the barcode is used to identify one or moremacromolecular constituents of the cell bead. In some cases, the barcodeis subjected to nucleic acid sequencing to identify one or moremacromolecular components. In some cases, the sequencing is untargetedsequencing. In some cases, the sequencing is targeted sequencing.

In operation 1595, the barcoded macromolecules (or derivatives thereof)are subjected to sequencing to generate reads. The sequencing may beperformed within a second droplet. The sequencing may be performedoutside of a second droplet. For instance, the sequencing may beperformed by releasing the barcoded macromolecules from a second dropletand sequencing the barcoded macromolecules using a sequencer, such as anIllumina sequencer or any other sequencer described herein. In somecases, a given barcoded sequencing read can be used to identify the cellfrom which the barcoded sequencing read was generated. Such capabilitycan link particular sequences to particular cells. Additional detailsand examples regarding nucleic acid sequencing methods are describedelsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 16 shows a droplet containing a single cell bead and a singlebarcode bead produced using the method 1500. An external droplet 1600 ofaqueous liquid is formed inside a first volume 1605 of a liquid that isimmiscible with the aqueous liquid. The external droplet contains asingle barcode bead 1620. Within the external droplet is an internaldroplet 1640 of aqueous liquid that comprises a cell bead. The internaldroplet is partitioned from the external droplet by a second volume 1630e of a liquid that is immiscible with the external droplet and internaldroplet. The internal droplet contains a single cell 1610, encapsulatedwithin the cell bead, containing one or more macromolecular constituents1615.

FIG. 17 shows a flowchart depicting an example method 1700 of producingdroplets containing a cell bead, in the form of a polymer-coated cell,and a barcode bead (e.g., a gel bead) and generating sequence reads frommacromolecular components of a cell associated with the cell bead. Insome cases, the method 1700 may comprise the following operations.

In operation 1710, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium.

In operation 1720, the cells can be exposed to a polymer thatselectively associates with the cells to form a coating on the cells.The polymer may be electrically charged. The polymer may comprise acation. The polymer may comprise a polycation. The coating may be formedby electrostatic interactions between the cells and the charged polymer.The polymer may be cholesterol. The polymer may be a lipid-modifiedcopolymer. The coating may be formed by hydrophobic interactions betweenthe cells and the polymer. The polymer may be a protein-modifiedcopolymer. The coating may be formed by protein interactions betweensurface antigens of the cells and the protein-modified copolymer. Thecoating may comprise one or more layers of coating. The coating may bediffusively permeable to chemical or biochemical reagents. The coatingmay be diffusively impermeable to macromolecular constituents of thecells. In this manner, the coating may act to allow the coated cells tobe subjected to chemical or biochemical operations while spatiallyconfining the macromolecular constituents to a region encapsulated bythe coating. The coating may comprise any other polymer capable ofinteracting with the cells.

In operation 1730, the coated cells can be subjected to conditionssufficient to lyse the cells. In some examples, lysis is completed withthe aid of a lysis agent in a droplet. In some cases, lysis of coatedcells is completed in bulk. The lysis of the cells may occur subsequentto subjecting the cells to conditions sufficient to encapsulate thecells in the polymer coating. The lysis may release macromolecularconstituents of the lysed coated cells. Though, the coating of the cellsmay retain the macromolecular constituents released from the cellswithin the confines of the coating. The lysis may be achieved byexposing the coated cells to sodium hydroxide (NaOH), potassiumhydroxide (KOH), or any other alkaline agent. The lysis may be achievedby exposing the coated cells to a detergent, such as sodium dodecylsulfate (SDS), 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol(TRITON X-100) or any non-ionic surfactant, or a saponin. The lysis maybe achieved by exposing the coated cells to an enzyme, such as aproteinase or a lytic enzyme (such as a lysozyme, cellulose, orzymolase). The lysis may be achieved by exposing the coated cells tofreeze thawing. The lysis may be achieved by exposing the coated cellsto electromagnetic radiation, such as ultraviolet (UV) light. The lysismay be achieved by exposing the coated cells to heat. The lysis may beachieved by exposing the coated cells to any other lysis agent. Thelysis may occur subsequent to forming a coating of the coated cells.

In operation 1740, the lysed coated cells can be subjected to conditionssufficient to denature one or more macromolecular constituents releasedby the lysed coated cells. In some examples, denaturation is completedwith the aid of a denaturation agent in a droplet. In some cases,denaturation is completed in bulk. The denaturing may be achieved byexposing the coated cells to sodium hydroxide (NaOH). The denaturing maybe achieved by exposing the coated cells to any other denaturing agent.In some examples, operation 1740 is completed contemporaneous tooperation 1730. In some examples, a denaturing agent can both denaturemacromolecular constituents and lyse the coated cells.

In operation 1750, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.The second liquid phase may comprise a cellular growth medium. Thesecond liquid phase may comprise a minimal growth medium. The barcodebeads each contain a barcode to barcode one or more macromolecularconstituents of the plurality of coated cells. In some cases, the firstliquid phase and the second liquid phase are the same phase. In somecases, the first liquid phase and the second liquid phase are mixed toprovide a mixed phase.

In operation 1760, the first liquid phase and the second liquid phaseare brought together with a third liquid phase that is immiscible withthe first and second liquid phase. The third liquid phase may interactwith the first and second liquid phases in such a manner as to partitioneach of the plurality of coated cells and the plurality of barcode beadsinto a plurality of droplets. The third liquid phase may comprise an oiland may also comprise a surfactant. The third liquid phase may comprisea fluorinated hydrocarbon. In some cases, a given droplet may include asingle coated cell and a single barcode bead. In some cases, at least80%, at least 90%, at least 95%, at least 99%, at least 99.5%, at least99.9%, at least 99.95%, or at least 99.99% of the droplets may contain asingle coated cell.

In operation 1770 and in the droplets, the coated cells are subjected toconditions sufficient to depolymerize the coating. The depolymerizationof the coating may be achieved by exposing the coated cells to areducing agent (e.g., dithiothreitol (DTT)), which may be in apartition. The depolymerization of the coating may be achieved byexposing the coated cells to any substance capable of depolymerizing thecoating. In some cases, operation 1770 also includes releasing barcodesfrom the barcode beads, which may be achieved with the same stimulus,such as, for example, used to reverse cross-linking of the coated cell.In some cases, the stimuli are different. Released barcodes can thenparticipate in barcoding as in operation 1780.

In operation 1780, the barcode is used to barcode one or moremacromolecular constituents of a given cell in a given droplet. In somecases, the macromolecular constituents of the cell is subjected toconditions sufficient for nucleic acid amplification for barcoding. Insuch cases, a barcode can function as a primer in such amplification. Inother cases, ligation can be used for barcoding. In some cases, thebarcode is used to identify one or more macromolecular constituents ofthe cell. In some cases, the barcode is subjected to nucleic acidsequencing to identify one or more macromolecular components. In somecases, the sequencing is untargeted sequencing. In some cases, thesequencing is targeted sequencing.

In operation 1790, the barcoded macromolecules (or derivatives thereof)are subjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell from which the barcodedsequencing read was generated. Such capability can link particularsequences to particular cells. Additional details and examples regardingnucleic acid sequencing methods are described elsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets.

FIG. 18 shows a droplet containing a single coated cell and a singlebarcode bead produced using the method 1700. A droplet 1800 of aqueousliquid is formed inside a volume 1805 of a liquid that is immisciblewith the aqueous liquid. The droplet contains a single barcode bead1820. The droplet also contains a single coated cell 1810 containing oneor more macromolecular constituents 1815. The coated cell is surroundedby a coating 1830 f.

FIG. 19 shows a flowchart that depicts an example method 1900 ofproducing droplets containing cell and a single barcode bead andgenerating sequence reads from macromolecular components of the cell. Inthis example, a droplet comprising aqueous fluids having differentviscosities can segregate an included cell to particular regions withinthe droplet. In this example, two miscible phases are provided in thedroplet, but which two miscible phases are of sufficiently differentphysical properties (e.g., have substantially different viscosities)that diffusion between the two phases is limited. In some examples, thetwo miscible phases are phases of an aqueous two phase system (ATPS).Examples of such two miscible phases include an aqueous phase and anaqueous phase comprising one or more of glycerol, ficoll, dextran andpolyethylene glycol (PEG). In this manner, incompatible chemical orbiochemical reagents may be sequestered into the different phases.Additionally, the slowed diffusion may allow for the timed exposure ofthe cell in the droplet or barcode bead to chemical or biochemicalreagents. In some cases, the method 1900 may comprise the followingoperations.

In operation 1910, a first liquid phase comprising a plurality of cellsis provided. The first liquid phase may be aqueous. The first liquidphase may comprise a cellular growth medium. The first liquid phase maycomprise a minimal growth medium. In some examples, the first liquidphase may comprise one of two miscible liquid phases between which twoliquid phases diffusion of molecules from one phase to the other islimited. For example the first liquid phase may comprise one componentof an ATPS or may comprise one or more viscosity enhancing agents, suchas glycerol, ficoll, dextran or polyethylene glycol (PEG).

In operation 1920, a second liquid phase comprising a plurality ofbarcode beads can be provided. The second liquid phase may be aqueous.In some examples, the second liquid phase may be the other component ofthe ATPS described above or may not include a viscosity enhancing agent,such as glycerol, ficoll, dextran and polyethylene glycol (PEG). Thesecond liquid phase may comprise a cellular growth medium. The secondliquid phase may comprise a minimal growth medium. The barcode beadseach contain a barcode to barcode one or more macromolecularconstituents of the plurality of cells.

In operation 1930, the first liquid phase and the second liquid phaseare brought together with a third liquid phase that is immiscible withthe first and second liquid phase. The third liquid phase may interactwith the first and second liquid phases in such a manner as to partitioneach of the plurality of cells and the plurality of barcode beads into aplurality of droplets. The third liquid phase may comprise an oil andmay comprise a surfactant. The third liquid phase may comprise afluorinated hydrocarbon. In some cases, a given droplet may include asingle cells and a single barcode bead. In some cases, at least 80%, atleast 90%, at least 95%, at least 99%, at least 99.5%, at least 99.9%,at least 99.95%, or at least 99.99% of the droplets may contain a singlecell.

In operation 1940, the cells are subjected to conditions sufficient tolyse the cells. In some examples, lysis is achieved via the aid of alysis agent present in the first liquid phase within a droplet. Thelysis may release macromolecular constituents of the lysed cells.However, given the difference in viscosities between the two fluids ofthe droplets, diffusion of these macromolecular constituents may belimited. The lysis may be achieved by exposing the cells to sodiumhydroxide (NaOH), potassium hydroxide (KOH), or any other alkalineagent, which may be in the droplet. The lysis may be achieved byexposing the cells to a detergent, such as sodium dodecyl sulfate (SDS),4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100) orany non-ionic surfactant, or a saponin, which may be in the droplet. Thelysis may be achieved by exposing the cells to an enzyme, such as aproteinase or a lytic enzyme (such as a lysozyme, cellulose, orzymolase), which may be in the droplet. The lysis may be achieved byexposing the cells to freeze thawing. The lysis may be achieved byexposing the cells to electromagnetic radiation, such as ultraviolet(UV) light. The lysis may be achieved by exposing the cells to heat. Thelysis may be achieved by exposing the cells to any other lysis agent.

In operation 1950, the lysed cells can be subjected to conditionssufficient to denature one or more macromolecular constituents releasedby the lysed cells. In some cases, denaturation is completed via the aidof a denaturation agent present in the first liquid phase within adroplet. The denaturing may be achieved by exposing the cells to sodiumhydroxide (NaOH), which may be in the droplet. The denaturing may beachieved by exposing the cells to any other denaturing agent, which maybe in the droplet. In some examples, operation 1950 is completedcontemporaneous to operation 1940. In some examples, a denaturing agentcan both denature macromolecular constituents and lyse the cells.

In operation 1960, the barcodes are used to barcode one or moremacromolecular constituents of a given cell in a given droplet.Barcoding can be timed by the limited diffusion of the macromolecularconstituents between the two phases within the droplet. After sufficienttime has passed for mixing of the macromolecular constituents withbarcode beads, barcoding can proceed. In some cases, the macromolecularconstituents are subjected to conditions sufficient for nucleic acidamplification for barcoding. In such cases, a barcode can function as aprimer in such amplification. In other cases, ligation may be used forbarcoding. In some cases, barcodes are used to identify one or moremacromolecular constituents of the cells. In some cases, barcodes aresubjected to nucleic acid sequencing to identify one or moremacromolecular components. In some cases, the sequencing is untargetedsequencing. In some cases, the sequencing is targeted sequencing. Insome cases, operation 1960 also includes releasing barcodes from thebarcode beads, which may be achieved with a stimulus such as a reducingagent (e.g. DTT). Released barcodes can then participate in barcoding.

In operation 1970, the barcoded macromolecules (or derivatives thereof)are subjected to sequencing to generate reads. The sequencing may beperformed within a droplet. The sequencing may be performed outside of adroplet. For instance, the sequencing may be performed by releasing thebarcoded macromolecules from a droplet and sequencing the barcodedmacromolecules using a sequencer, such as an Illumina sequencer or anyother sequencer described herein. In some cases, a given barcodedsequencing read can be used to identify the cell from which the barcodedsequencing read was generated. Such capability can link particularsequences to particular cells. Additional details and examples regardingnucleic acid sequencing methods are described elsewhere herein.

In some cases, prior to sequencing, the barcoded macromolecules may befurther processed. For example, the barcoded macromolecules aresubjected to nucleic acid amplification (e.g., PCR) prior to sequencing.In some cases, additional sequences are ligated to barcodedmacromolecules. Such further processing may be performed in a droplet orexternal to the droplet, such as by releasing the barcodedmacromolecules from the droplets. FIG. 20 shows a droplet containing asingle cell and a single barcode bead produced using the method 2000. Adroplet 2000 of aqueous liquid is formed inside a volume 2005 of aliquid that is immiscible with the aqueous liquid. The droplet containsa single gel bead 2020. The droplet also contains a single cell 2010containing one or more macromolecular constituents 2015. The dropletalso contains two different aqueous phases that separately confine thebarcode bead 2020 and the cell 2010. Phase 2030 g comprises the cell2010 and phase 2030 a comprises the barcode bead 2020.

The disclosure also provides compositions, systems and methods forgenerating cell beads in cell beads. Such methods, compositions andsystems can be useful for positioning cells encapsulated in cell beadsat the center or substantially at the center of the cells beads. In somecases, centering of a cell can prevent the contents of the cell beads(e.g., cells, components of cells, biomolecules derived from cells,nucleic acids from cells) from diffusing or leaking out of the cellbead. Loss of these materials can lead to partial or complete loss ofthe sequencing information for the contents of a given cell bead. Forexample, leakage of nucleic acids from cells at the edges of cell beadscan lead to noisy profiles derived from sequencing and/or potentialfalse positive calls. By centering cells within cell beads, a greaterdepth of cell bead material encapsulates cells, providing a largerdiffusion distance and, thus, greater diffusion barrier for diffusion ofencapsulated materials. Moreover, a cell bead in cell bead approach,itself, adds additional material that surrounds the cell, also resultingin a greater diffusion barrier. In general, cell beads in cell beads canbe generated by a similar process used to generate single gel beads, asdescribed elsewhere herein. First order cell beads can be generated asdescribed herein, and then subjected to the same process for cell beadgeneration again to generate cell beads in cell beads.

An example method and microfluidic device architecture for generatingcell beads in cell beads are schematically depicted in FIG. 27. As shownin FIG. 27, cell beads 2701, which contain cells 2702 may be generatedin any suitable manner, including in a manner described herein, areprovided in an aqueous phase. The cell beads 2701 are then provided 2703to a microfluidic device 2704. The device comprises microfluidicchannels arranged in a double-cross configuration. The cell beads 2701are provided to the microfluidic device where they flow in a firstchannel 2705 of the microfluidic device 2704 to a first channelintersection with second and third channels 2706 and 2707. The secondand third channels 2706 and 2707 provide polymeric or gel precursorsthat come together with the stream of cell beads 2701 from the firstmicrofluidic channel 2705.

The stream comprising the cell beads 2701 and polymeric or gelprecursors then flows through a fourth microfluidic channel 2708 to asecond channel intersection with fifth and sixth channels 2709 and 2710.The fifth and sixth channels provide a phase immiscible with the aqueousphase of cell beads 2701 and polymeric or gel precursors flowing inchannel 2708. The stream comprising the cell beads 2701 and polymeric orgel precursors from the fourth channel 2708 flows into the immisciblestream such that droplets 2711 comprising cell beads and polymeric orgel precursors are generated and flow away from the second intersectionin a seventh channel 2712. The droplets 2711 can then be subject toconditions suitable for polymerizing or gelling the precursors in thedroplets 2711 and subject to solvent exchange as is described elsewhereherein and the resulting cell beads in cell beads recovered.

A photograph showing generation of droplets comprising cell beads andpolymeric or gel precursors using a microfluidic device, similar to thatshown schematically in FIG. 27, is shown in FIG. 28A. As shown anaqueous phase comprising cell beads 2701 provided from channel 2705 isprovided to a first channel junction, into which aqueous phase polymericor gel precursors flow from channel 2706. The resulting aqueous mixture,comprising both cell beads 2701 and polymeric or gel precursors, flowsthrough channel 2708 into a second channel junction, into which oilprovided by channel 2709 flows. The interaction between oil and aqueousphases generates droplets 2711 that comprise a cell bead 2701 andpolymeric or gel precursors that flow away from the second channeljunction in channel 2712.

FIG. 28B shows a photograph of a cell bead in cell bead generated fromdroplets generated in FIG. 28A. The cell bead in cell bead comprises alarger cell bead 2800 that encapsulates a smaller cell bead 2801. Thesmaller cell bead 2801 encapsulates a cell 2802. As shown in FIG. 28B,the cell 2802 is substantially centered within the larger cell bead2800.

FIG. 29 shows a histogram of Depth Positional Coefficient of Variation(DPCV) values across individual cells, whose nucleic acids weresequenced using a cell bead in cell bead sample preparation approach.DPCV is a measure of the evenness of sequencing coverage achieved acrossthe position of the genome.

Additionally, cells may be centered in droplets without the generationof a cell bead comprising a cell bead. For example, droplets comprisingpolymeric or gel precursors and cells may be subjected to shearing priorto cell bead generation. Shearing may be achieved, for example, viaorbital shaking or in a microfluidic channel. In such cases, thekinetics of polymerization or gelation of the precursors can becontrolled such that polymerization or gelation is sufficiently slow ordelayed. Slower or delayed polymerization or gelling can permit internalcirculation of droplet contents that can center a cell within a droplet,such that it can then be fixed in place at the center of a cell beadupon precursor polymerization or gelling.

FIG. 30 depicts a bar plot showing different categories of cell beads asa function of example conditions used to make the cell beads. The cellbeads were classified into three categories (edge, off-center, center)depending on the location of a cells with respect to either the edge orthe center of a given cell bead. The cell beads were generated withvarying time and speed of shaking on an orbital shaker, as is discussedabove.

Furthermore, cells may also be centered in droplets by formingcore-shell beads, with cells suspended in the solution that forms thecore. Cells may be formed by viscosity-mismatched flowing streams suchthat cells are suspended in a core fluid having a different viscositythan a shell fluid. The shell fluid may be liquid and/or formed from across-linked matrix such as a cross-linked polymer. Examples of suchcore-shell beads are described in Rossow et al., J. Am. Chem. Soc. 2012,134, 4983-4989, which is incorporated herein by reference.

Core-shell beads having cells suspended in the cores may also be formedthrough the generation of aqueous-in-aqueous droplets made from aqueoustwo-phase systems. For example, the cells are suspended in a coresolution (e.g., a polymer core solution, a polyethylene glycol (PEG)core solution) that is then surrounded by a cross-linked shell (e.g.,cross-linked dextran shell). This bead may be generated fromaqueous-in-aqueous droplets with one aqueous phase comprising cross-linkprecursors and another aqueous phase comprising cells. Additionaldetails regarding the formation of core-shell beads from aqueoustwo-phase systems are provided in Mytnyk et al., RSC Adv., 2017, 7,11331-11337, which is incorporated herein by reference.

Many variations, alterations and adaptations based on the disclosureprovided herein are possible. For example, the order of the operationsof one or more of the example methods 700, 900, 1000, 1100, 1300, 1500,1700, 1900, and 2500 discussed above can be changed, some of theoperations removed, some of the operations duplicated, and additionaloperations added as appropriate. Some of the operations can be performedin succession. Some of the operations can be performed in parallel. Someof the operations can be performed once. Some of the operations can beperformed more than once. Some of the operations can comprisesub-operations. Some of the operations can be automated and some of theoperations can be manual. The processor as described herein can compriseone or more instructions to perform at least a portion of one or moreoperations of one or more of the methods. Moreover, while these examplesare described above with respect to cell analysis, the same procedurescan be extended to other biological species containing macromolecularconstituents that can be barcoded, including viruses.

Also disclosed herein are systems for cell analysis, including via acell bead. The systems may utilize a droplet generator (e.g., amicrofluidic device, droplet generators having a T-junction, dropletgenerators that generate droplets with cross-channel flow focusing,droplet generators that generate droplets with step/edge emulsification,droplet generations that generate droplets with gradient generation,droplet generators that use piezo/acoustics for droplet generation).

In some cases, a droplet generator is a microfluidic device whichincludes mixing of immiscible fluids at channel junctions of one or morechannels to form droplets. The channels may be microchannels. Themicrochannels may be implemented on microfluidic devices. Examples ofsuch microfluidic devices and their operation are provided in FIG. 1B,FIG. 1C, FIG. 1D, and FIG. 31 and are described elsewhere herein.

Such systems may also include a controller programmed to implement amethod described herein, including one of the example methods 100, 700,900, 1000, 1100, 1300, 1500, 1700, 1900, and 2500 described herein.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 6 shows acomputer system 601 that is programmed or otherwise configured toimplement methods or parts of methods described herein, includingexample methods 100, 700, 900, 1000, 1100, 1300, 1500, 1700, 1900, and2500. The computer system 601 can regulate various aspects of thepresent disclosure, such as, for example, sample preparation of cellularmaterials in cell beads, barcoding of these materials and/or analysis ofbarcoded molecules. The computer system 601 can be an electronic deviceof a user or a computer system that is remotely located with respect tothe electronic device. The electronic device can be a mobile electronicdevice.

The computer system 601 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 605, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 601 also includes memory or memorylocation 610 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 615 (e.g., hard disk), communicationinterface 620 (e.g., network adapter) for communicating with one or moreother systems, and peripheral devices 625, such as cache, other memory,data storage and/or electronic display adapters. The memory 610, storageunit 615, interface 620 and peripheral devices 625 are in communicationwith the CPU 605 through a communication bus (solid lines), such as amotherboard. The storage unit 615 can be a data storage unit (or datarepository) for storing data. The computer system 601 can be operativelycoupled to a computer network (“network”) 630 with the aid of thecommunication interface 620. The network 630 can be the Internet, aninternet and/or extranet, or an intranet and/or extranet that is incommunication with the Internet. The network 630 in some cases is atelecommunication and/or data network. The network 630 can include oneor more computer servers, which can enable distributed computing, suchas cloud computing. The network 630, in some cases with the aid of thecomputer system 601, can implement a peer-to-peer network, which mayenable devices coupled to the computer system 601 to behave as a clientor a server.

The CPU 605 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 610. The instructionscan be directed to the CPU 605, which can subsequently program orotherwise configure the CPU 605 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 605 can includefetch, decode, execute, and writeback.

The CPU 605 can be part of a circuit, such as an integrated circuit. Oneor more other components of the system 601 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 615 can store files, such as drivers, libraries andsaved programs. The storage unit 615 can store user data, e.g., userpreferences and user programs. The computer system 601 in some cases caninclude one or more additional data storage units that are external tothe computer system 601, such as located on a remote server that is incommunication with the computer system 601 through an intranet or theInternet.

The computer system 601 can communicate with one or more remote computersystems through the network 630. For instance, the computer system 601can communicate with a remote computer system of a user. Examples ofremote computer systems include personal computers (e.g., portable PC),slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab),telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device,Blackberry®), or personal digital assistants. The user can access thecomputer system 601 via the network 630.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 601, such as, for example, on the memory610 or electronic storage unit 615. The machine executable or machinereadable code can be provided in the form of software. During use, thecode can be executed by the processor 605. In some cases, the code canbe retrieved from the storage unit 615 and stored on the memory 610 forready access by the processor 605. In some situations, the electronicstorage unit 615 can be precluded, and machine-executable instructionsare stored on memory 610.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 601, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 601 can include or be in communication with anelectronic display 635 that comprises a user interface (UI) 640.Examples of UI's include, without limitation, a graphical user interface(GUI) and web-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 605. Thealgorithm can, for example, implement methods or parts of methodsdescribed herein, including example methods 100, 700, 900, 1000, 1100,1300, 1500, 1700, 1900, and 2500.

EXAMPLES Example 1 Detection of Infectious Agents

The systems and methods described herein may be used to detectinfectious agents in cells. For instance, the systems and methods may beused to collect CD4 T-cells in droplets and subject the collected CD4T-cells to nucleic acid sequencing. For CD4 T-cells obtained from anHIV-infected subject, the nucleic acid sequencing may reveal thepresence of HIV-derived nucleic acids in the cells. The extent to whichthe HIV infection has spread in an HIV-infected subject may be measuredby collecting and performing nucleic acid sequencing on all CD4 T-cellsobtained from the HIV-infected subject. The systems and methods may beused to detect any infections agents in cells.

The systems and methods may be used to detect co-infections by two ormore infectious agents in cells. In an example, a subject's cells may becollected in droplets and subjected to nucleic acid sequencing. Thenucleic acid sequencing may reveal the presence of two or moreinfectious agent-derived nucleic acids in the cells. Alternatively or incombination, the cells collected in droplets may be subjected to anantibody-based multiple assay. The multiple assay may reveal thepresence of two or more infectious agents.

Example 2 Preparation of Long DNA Reads

The systems and methods described herein may be utilized to retain longnucleic acid segments for producing long sequencing reads while removingshort nucleic acid segments. The retention of long nucleic acid segmentsand removal of short nucleic acid segments may enhance the accuracy orspeed of nucleic acid sequencing technologies, such as those nucleicacid sequencing technologies described herein.

FIG. 23 shows a schematic depicting an example method 2300 for retaininglong nucleic acid segments and removing short nucleic acid segments.

In a first operation 2310, a mixture of short and long DNA segments iscollected.

In a second operation 2320, the mixture of long and short DNA segmentsis loaded into cell beads. The mixture may be loaded into the cell beadsby any of the systems and methods described herein. The mixture may beloaded into the cell beads such that some cell beads enclose a mixtureof short nucleic acid segments and long nucleic acid segments.

In a third operation 2330, the cell beads are washed. During washing,the short nucleic acid segments are washed out of the cell beads, suchthat the cell beads retain the long nucleic acid segments. Cell beadscan be tailored to have porosity that traps longer nucleic acid segmentswithin cell beads but allows shorter nucleic acid segments to diffuse orflow out of the cell beads.

In a fourth operation 2340, the cell beads containing long nucleic acidsegments are combined with gel beads to form droplets containing one ormore cell beads and one or more gel beads.

In a fifth operation 2350, the DNA segments are subjected to nucleicacid sequencing, as described herein.

Although described herein with respect to nucleic acids, the method 2300may be used to generate droplets containing long segments of anymacromolecules described herein. For instance, the method 2300 may beused to generate droplets containing long protein segments.

Example 3 Amplification of Specific Nucleic Acid Loci

The systems and methods described herein may be used in theamplification and barcoding of targeted sequences, such as nucleic acid(e.g. DNA) loci. These nucleic loci may be derived from nucleic acidsthat are associated with or encapsulated within a cell bead. Moreover,amplification may be performed in an individual partition among aplurality of partitions, such as a droplet among a plurality ofdroplets. Where partitions are implemented, an individual partition maycomprise a cell bead having the nucleic acid(s) to be amplified. In somecases, amplification of nucleic acid(s) of a cell bead may be completedprior to partitioning. FIG. 24 shows an example process for amplifyingand barcoding targeted nucleic acid sequences.

During a first stage of amplification, the forward primers hybridizewith their respective loci where present and are extended via the actionof the polymerase and, in some cases, with the aid of thermal cycling.The resulting constructs (not shown in FIG. 24) comprise both theuniversal nucleic acid sequence and complementary sequences of targetloci present. In a second stage of amplification, the reverse primershybridize to the complementary sequences of the target loci generated inthe first stage and are extended to generate constructs (not shown inFIG. 24) comprising the original loci sequences and a complementarysequence of the universal nucleic acid sequence. In some cases, theconstructs generated in the second stage are shorter in length thanthose generated in the first round, such that the sequences derived fromthe nucleic acids analyzed that are present in these constructs are thetarget loci sequences.

Next, barcoded nucleic acid molecules, shown in FIG. 24, comprising anR1 primer sequence (e.g., primer for sequencing), a barcode sequence(BC), and the universal nucleic acid sequence are provided. The barcodednucleic acid molecules may be coupled to beads and/or may be releasablefrom the beads. In some cases, these beads are partitioned with cellbeads in which amplification of target loci has already been completedprior to partitioning. In other cases, these beads are partitioned withcell beads prior to such amplification. Moreover, where releasable frombeads, the barcoded nucleic acid molecules can be released from thebeads prior to participating in further downstream reactions.

The barcoded nucleic acid molecules can be contacted with the amplifiednucleic acids generated above and corresponding to the various locipresent. Upon contact, the universal nucleic acid sequence of thebarcoded nucleic acid molecules can hybridize with complementarysequences generated in second-stage constructs discussed above. Thehybridized barcoded nucleic acid molecules are then extended via theaction of a polymerase, such as with the aid of thermal cycling, togenerate barcoded constructs comprising the sequences of the barcodednucleic acid molecules and sequences complementary to the second stageconstructs discussed above and corresponding to the original locisequences analyzed. In some cases, the resulting barcoded constructs canthen be further processed to add additional sequences and then subjectto sequencing. As shown, amplification schemes described above cangenerate barcoded, target-specific constructs for sequencing analysis.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1-30. (canceled)
 31. A method for nucleic acid sequencing, comprising:(a) providing a cell bead comprising a cell or constituent of said cell,wherein said cell or constituent comprises a nucleic acid molecule; (b)within said cell bead, processing said nucleic acid molecule with areagent to generate a modified nucleic acid molecule in said cell bead,which modified nucleic acid molecule comprises one or more modifiednucleic acid bases that are modified with respect to said nucleic acidmolecule; and (c) subjecting said modified nucleic acid molecule or aderivative thereof to sequencing to identify a sequence comprising saidone or more modified nucleic acid bases.
 32. The method of claim 31,wherein said nucleic acid molecule is subject to a modification selectedfrom the group consisting of incorporation of one or more modified ornon-canonical bases, digestion, methylation, random mutagenesis,bisulfite conversion, uracil hydrolysis, nucleic acid repair,ribonucleic acid (RNA) capping, RNA decapping, nucleic acid insertion,and nucleic acid cleavage of or to said nucleic acid molecule togenerate said modified nucleic acid molecule.
 33. The method of claim31, wherein (a) comprises providing a plurality of cell beads includingsaid cell bead, and (b) comprises subjecting said plurality of cellbeads to processing in bulk together with other cell beads of saidplurality of cell beads, wherein said processing comprises modifyingsaid one or more nucleic acid bases of said nucleic acid molecule withinsaid plurality of cell beads.
 34. The method of claim 31, furthercomprising providing a nucleic acid barcode molecule comprising abarcode sequence; and using said nucleic acid barcode molecule and saidmodified nucleic acid molecule or derivative thereof to generate abarcoded nucleic acid molecule comprising (i) a sequence of saidmodified nucleic acid molecule, or a reverse complement thereof, and(ii) said barcode sequence, or a reverse complement thereof.
 35. Themethod of claim 34, further comprising providing a barcode bead, whereinsaid barcode bead comprises said nucleic acid barcode molecule coupledthereto.
 36. The method of claim 35, wherein said barcode bead comprisesa plurality of nucleic acid barcode molecules including said nucleicacid barcode molecule, and wherein said plurality of nucleic acidbarcode molecules comprises said barcode sequence.
 37. The method ofclaim 35, wherein said barcode bead comprises a polymer or gel matrix.38. The method of claim 35, further comprising releasing said nucleicacid barcode molecule from said barcode bead upon application of one ormore stimuli.
 39. The method of claim 31, further comprising, prior to(c), partitioning said cell bead in a partition.
 40. The method of claim39, wherein said partition is a droplet or a well.
 41. The method ofclaim 39, further comprising, prior to (c), (i) co-partitioning saidcell bead and a barcode bead into said partition, wherein said barcodebead comprises a nucleic acid barcode molecule coupled thereto, whereinsaid nucleic acid barcode molecule comprises a barcode sequence; and(ii) using said nucleic acid barcode molecule and said modified nucleicacid molecule or derivative thereof to generate a barcoded nucleic acidmolecule comprising (1) a sequence of said modified nucleic acidmolecule, or a reverse complement thereof, and (2) said barcodesequence, or a reverse complement thereof.
 42. The method of claim 41,further comprising releasing said modified nucleic acid molecule fromsaid cell bead into said partition upon application of a stimulus. 43.The method of claim 41, wherein said barcode bead comprises a pluralityof nucleic acid barcode molecules including said nucleic acid barcodemolecule, and wherein said plurality of nucleic acid barcode moleculescomprises said barcode sequence.
 44. The method of claim 41, furthercomprising, prior to (c), recovering said barcoded nucleic acid moleculeor derivative thereof from said partition.
 45. The method of claim 31,further comprising performing one or more reactions on said modifiednucleic acid molecule prior to said sequencing in (c).
 46. The method ofclaim 45, wherein said one or more reactions comprise polymerase chainreaction (PCR).
 47. The method of claim 45, wherein said one or morereactions comprise nucleic acid amplification.
 48. The method of claim31, wherein said cell bead comprises a polymer or gel matrixencapsulating said cell or said constituent of said cell.
 49. The methodof claim 48, wherein said polymer or gel matrix is degradable uponapplication of one or more stimuli selected from the group consisting oftemperature change, pH change, environmental change, exposure to achemical species, exposure to a chemical phase, and exposure to light.50. The method of claim 49, further comprising releasing said modifiednucleic acid molecule from said cell bead upon application of saidstimulus.